Purification and partial characterization of plasminogen activator from human uterine tissue.

A procedure was developed for the purification of a plasminogen activator from human uterine tissue. It involves six consecutive steps: (1) extraction of the plasminogen activator from delipidated uterine tissue with 0.3 M potassium acetate buffer, pH 4.2; (2) ammonium sulphate precipitation; (3) zinc chelate-agarose chromatography; (4) n-butyl-agarose chromatography; (5) concanavalin A-agarose chromatography; and (6) gel filtration on Sephadex G-150. The specific activity of the final plasminogen activator preparation was increased by a factor 4500 as compared with the crude extract. The purified plasminogen activator showed a strong tendency to adsorb to surfaces. This could be effectively prevented by Tween-80. The molecular weight of the plasminogen activator was 64 000 as estimated by gel filtration in 1.0 M NaCl and 69 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The plasminogen activator consisted of two chains (molecular weights 31 000 and 38 000) connected by disulphide bridges. The smallest chain contained the serine residue of the active site as deduced from the incorporation of the tritium label of [3H]diisopropylphosphofluoridate.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

Isolation and purification of cyanogen bromide-derived peptides of type II collagen directly from tissue (Swarm chondrosarcoma)

A preparative procedure is described for isolating type II collagen-fragments directly from tissue. Swarm chondrosarcoma from rat, a cartilagenous tissue rich in type II collagen, was digested by cyanogen bromide in 70% formic acid. The resulting crude extract was desalted (G 25 column chromatography) and lyophylized. The yield of peptide mixture was about 1 250 mg obtained from 100 g tissue. The method of purification commonly used for type II collagen prior to cyanogen bromide-cleavage yielded 20 mg peptides from 100 g tissue. Separation of the cyanogen bromide-derived fragments was performed by gel filtration. The column was run at 43 degrees C (denaturing-temperature of collagens) to avoid fibril formation, and a volatile buffer was used (ammonium formate buffer, pH 7.5) so that the effluent fractions could be easily lyophylized. Two-dimensional gel electrophoresis of the main peaks of the column profile demonstrated that this purification step resulted in a good separation of the fragment mixture, although additional steps may be necessary for complete separation of the peptides. The most striking advantages of the method for direct digestion of tissue outlined here are the increase in yield (about 60-fold) and the reduction of purification steps (avoiding type II collagen purification).     

The partial purification and properties of pepsin obtained from Turkey proventriculus

In this study, pepsin from turkey proventriculus was purified, and its biochemical properties examined. Initially, the turkey proventriculus (stomach) was mixed with 10% NaCl (1∶2, w/v) and extracted by centrifugation to produce a crude extract. The partial purification of the extract was carried out using Sephadex G-50 resin in gel filtration column chromatography. The fractions obtained by gel filtration were analyzed for milk clotting activity (MCA), protein content, proteolytic activity (PA), purification factor (PF), and SDS-PAGE electrophoresis was also performed. The enzyme was purified 207-fold with a recovery of 36%. The first 4 fractions did not have any activities; fractions 7, 8, and 9 exhibited the highest levels of milk clotting and proteolytic activity. The electrophoretic patterns revealed that further purification steps should be applied for better results.     

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.     

Two novel antimicrobial peptides purified from the symbiotic bacteria Xenorhabdus budapestensis NMC-10

Symbiotic bacteria, which are carried in the intestinal vesicle of the infective stage of juvenile entomopathogenic nematodes, produce broad-spectrum antibiotics. In this study, we aimed to isolate the antimicrobial peptides from the culture of the entomopathogenic bacterium Xenorhabdus budapestensis NMC-10. By screening chromatography columns and optimizing flow rate, pH, salinity and other purification conditions, we identified the final purification procedures which consisted of Q ion-exchange chromatography, gel filtration chromatography and two-step reverse-phase chromatography. Two novel antimicrobial peptides were identified via Q-TOF-TOF and de novo sequencing, and designated as GP-19 and EP-20. Both natural and synthetic peptides demonstrated broad-spectrum antimicrobial activities. The synthetic GP-19 peptide was active against Verticillium dahlia with EC(50) values of 17.54 μg/ml and highly inhibited the growth of a variety of bacteria, while the synthetic EP-20 peptide was highly active against Phytophthora capsici with EC(50) values of 3.14 μg/ml.     

Detergent effect on anion exchange perfusion chromatography and gel filtration of intact chloroplast H(+)-ATP synthase

To gain a pure enzyme preparation for functional and crystallization studies, an additional purification step in the isolation of the chloroplast ATP synthase (CF(0)F(1)) has been introduced. By applying gel filtration or anion exchange perfusion chromatography in presence of the detergents CHAPS and n-dodecyl-beta-d-maltoside, respectively, Rubisco and other contaminants were separated from CF(0)F(1). The purity and activity depended on the chromatographic method and the detergent employed. The highest purity and activity were achieved by anion exchange chromatography for the detergent dodecyl-maltoside and by gel filtration for the detergent CHAPS. The detergent Triton X-100, which is frequently used to solubilize CF(0)F(1), was found to be inadequate to stabilize the ATP synthase during chromatography.     

Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae)

In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil elastase from normal donors

Human neutrophil elastase from normal donors has been purified using an isolation procedure which included sequential sodium chloride extraction, Aprotinin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil elastase and resulted in a higher specific activity of the final preparation. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of the reduced purified protein demonstrated three polypeptides of Mr 31,000, 28,000, and 27,500. Four polypeptides were resolved on acid gel electrophoresis; each of the four possessed amidolytic activity. Furthermore, peptide analysis of Staphylococcus aureus V8 protease digests indicated that these polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The apparent isoelectric points of these four forms as determined by two-dimensional electrophoresis range from 6.1 to 6.7. By utilizing microsequencing techniques, the first 40 residues of neutrophil elastase have been determined and compared with the reported sequence of elastase isolated from leukemic myeloid cells. In addition, a high degree of homology was found within the amino-terminal regions of neutrophil elastase and the serine proteinases porcine elastase, bovine chymotrypsin, human factor D, and the beta chain of plasmin.     

Pleurostrin, an antifungal peptide from the oyster mushroom

A 7kDa peptide, with inhibitory activity on mycelial growth in the fungi Fusaerium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, was isolated from fresh fruiting bodies of the oyster mushroom. The isolation procedure entailed extraction with an aqueous buffer, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. It demonstrated an N-terminal sequence different from known antifungal proteins and peptides.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.     

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.     

Partial purification of pepsin from turkey proventriculus

In the present study, turkey proventriculus (stomach) was mixed with 10% NaCl (1:2 w/v) and extracted by centrifugation to produce crude extract. The partial purification of the extract was carried out by using Sephadex G-75 resin in gel filtration column chromatography. Crude extract (CE) and fractions obtained by gel filtration were analysed for milk-clotting activity (MCA), protein content, proteolytic activity (PA), and SDS-PAGE electrophoresis. The first 7 fractions did not have any activities; the fraction of 9, 10 and 11 had the most milk-clotting and proteolytic activities. Electrophoretic patterns proved that further purification steps should be applied for better results.     

Isolation and purification of a type-specific antigen from Chlamydia trachomatis propagated in cell culture utilizing molecular shift chromatography

Various techniques have been utilized for antigen solubilization, isolation, and purification. This report is the first to describe the isolation and purification of a type-specific antigen from Chlamydia trachomatis serotype A grown in cell culture. The type-specific antigen was prepared from Chlamydia trachomatis serotype A organisms grown in baby hamster kidney cells (BHK21). The extraction process employed a combination of both pH change and Triton X-100 solubilization. The soluble extract was radioiodinated and subjected to ion exchange and gel filtration chromatography. The fractions eluted were tested for type specificity utilizing the IgG prepared from exhaustively cross-absorbed hyperimmune sera from rabbits immunized with homologous organisms. Molecular shift chromatography was employed for analysis. Small samples of the isolated antigen were later used as markers for preparation of larger quantities necessary for antigenic characterization. The purified type-specific antigen has a m.w. of 30,000 to 32,000.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

Identification and Characterization of a Polypeptide from a Lobster Neurosecretory Gland that Induces Cyclic GMP Accumulation in Lobster Neuromuscular Preparations

Several observations suggest that cyclic GMP might regulate some aspect of neuromuscular physiology or metabolism in the lobster. Homarus americanus: lobster muscle is one of the richest known sources of cyclic GMP-dependent protein kinase, the preparation contains several phosphoproteins whose state of phosphorylation is affected by cyclic GMP more effectively than by cyclic AMP, and guanylate cyclase and phosphodiesterase are active in this tissue. However, no factor has yet been identified that alters lobster muscle cyclic GMP levels. We have screened extracts of neural and neurosecretory structures for the capacity to promote cyclic GMP accumulation in isolated exoskeletal muscles. Extracts of the sinus gland (a neurohemal organ found in the eyestalk) contain a factor that induces up to 100-fold increases in muscle cyclic GMP content, whereas extracts of other tissues are ineffective. This factor can also act on targets other than muscle, with hepatopancreas, testis, and neuronal tissue showing the largest responses. The sinus gland factor does not appear to affect cyclic GMP metabolism by depolarizing the preparation or by mobilizing extracellular Ca2+. The effect on cyclic GMP levels is dose-dependent and linear with time. Biological activity is destroyed by boiling and by 90% ethanol. It is also destroyed by trypsin, chymotrypsin, or pronase, which suggests that the factor is a protein or peptide. Both gel filtration chromatography and experiments using dialysis tubing with different molecular weight exclusion limits indicate that the factor has an apparent molecular weight of 5,000-12,000 daltons. A preliminary fractionation scheme, based on gel filtration, ion-exchange, and reverse-phase chromatography, gives greater than 1,300-fold purification. Our long-range goal is to purify this factor to homogeneity, compare it to other peptide hormones, and use it as a probe to evaluate the role of cyclic GMP at the neuromuscular junction.     

Modified simplified method for isolation of lysostaphin from the culture filtrate ofStaphylococcus staphylolyticus

The present paper reports a modified method for isolation of lysostaphin—a bacteriolytic agent with specific affinity for staphylococcal cell wall. The proposed purification scheme includes three steps. The first procedure is ultrafiltration through a membrane filter giving a yield of 75.6 %. The result of ultrafiltration is a concentrated, 10-times purified preparation of lysostaphin with specific activity 0.62 U/mg which can be used for digestion ofS. aureus cells. Further step, performed by ion-exchange chromatography on DEAE-cellulose, yields a 60-times purified preparation containing a mixture of enzyme components of lysostaphin. The yield of this step is 47.2 %, the preparation contains 3.54 U/mg protein. Using gel filtration on Sephadex G-50 a component with hexosaminidase activity was separated from the endopeptidase component on the basis of molar mass difference. A 270-times purified preparation of lysostaphin-endopeptidase with minimum of contaminating substances was obtained in this step. The yield of gel filtration was 22.1 %, specific activity increased up to 16.3 U/mg protein.     

一种猪溶菌酶来源的抗菌六肽的分离鉴定及其性质

为提高猪溶菌酶(Sus scrofa lysozyme,SSL)的抗革兰氏阴性菌活性,将其进行了不同蛋白酶的水解,选择抗革兰氏阴性菌效果最好的水解产物,利用凝胶过滤色谱和反相制备色谱进行分离,对其功能成分进行液质联用鉴定。对分离得到的物质进行抗菌活性验证和生物信息学的分析,并在此基础上对抗菌物质的杀菌机理进行了探讨。结果表明,胰蛋白酶的水解产物具有较高的杀灭革兰氏阴性菌的活性,进一步分离纯化得到了具有抗革兰氏阴性菌活性的六肽A-W-V-A-W-K。经化学合成验证,该六肽既保留了SSL的部分抗菌活性,也具备杀灭多种革兰氏阴性菌的能力。进一步分析发现其位于SSL分子C端的一个螺旋-回环-螺旋的结构中,并由此推测其杀菌机理是通过改变细胞膜的渗透性,进而使细胞内溶物流出而造成细胞死亡,而抗菌实验也验证了这一推测。该抗菌肽的发现为后续提高SSL的抗菌活性提供了理论依据。