Proteinase inhibitors from the tropical sea anemone Radianthus macrodactylus: Isolation and characteristic

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.     

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.     

High-performance liquid chromatography of flufenamic acid in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for the determination of flufenamic acid in rat plasma is described. After liquid-liquid extraction, the drug is separated by HPLC on a 5-μm octadecylsilica column (Nucleosil C₁₈) with ultraviolet detection at 280 nm. Linear calibration graphs for flufenamic acid were constructed from 0.5 to 15 μg/ml. The method has been applied to a pharmacokinetic study in animals.     

Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes

The isolation and structural characterization of fucosylatedneolacto-series gangliosides with linear poly N-acetyllactosaminylchains from normal human granulocytes is described. Gangliosideswere purified by consecutive use of anion exchange HPLC on FractogelTMAE-650(S), adsorption and reversed phase HPLC on Nucleosil50-7 and Nucleosil 7C₁₈ columns, respectively. TLC immunostainingwith carbohydrate specific monoclonal antibodies, fast atombombardment-mass spectrometry (FAB-MS) of the permethylatedderivatives and gas chromatography-electron impact mass spectrometry(GC-EIMS) of partially methylated alditol acetates were usedfor structure elucidations. One ganglioside was identified assialyl Lewis antigen with nLcOse₆Cer core, Neu5-Ac     

Isolation and separation of the glycan strands from murein of Escherichia coli by reversed-phase high-performance liquid chromatography

The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase. The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile. The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues. This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18. The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60. The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units.     

Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.     

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.     

Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons

A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na+/K+-ATPase activity and displacing ouabain were collected and purified further. By comparison with ouabain, the final extract was found to have a steeper concentration-effect curve in the inhibition of Na+/K+-ATPase. In displacement of [3H]ouabain, the extract had again a steeper concentration-effect curve than does ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72% of the total) and three saturated hydrocarbons. The assignment of structures was corroborated by comparison with authentic samples.     

Synthesis and properties of N-bromoacetyl-L-thyroxine.

A one-step bromoacetylation of L-thyroxine (T4) produces N-bromoacetyl-L-thyroxine (BrAcT4) in good yield. The reaction product is best purified by high-speed countercurrent chromatography. While HPLC is satisfactory only for purification of microgram and submicrogram quantities, amounts ranging from about 1 ng to 1 g of BrAcT4 can be processed by high-speed countercurrent chromatography (HSCCC), a method which we have previously used for the purification of N-bromoacetyl-3,3',5-triiodo-L-thyronine (BrAcT3). Operating conditions for the one-step synthesis of BrAcT4 and BrAcT3 differ due to differences in solubility and reactivity of the two hormones. BrAcT4 purified by HSCCC and shown to be pure by analytical HPLC has been characterized by alpha max and epsilon max in the near and far uv in several solvents, mass spectrum, 1H NMR spectrum, TLC in three solvent systems, retention time in reverse-phase HPLC (C18) in relation to the retention times of two internal standards, 3,3',5-triiodo-L-thyronine and T4, and melting point. Corresponding data for BrAcT3, not previously reported, have also been determined. The described procedure can provide not only substantial amounts of highly purified BrAcT4 for competition studies, but also 125I-labeled BrAcT4 of high specific activity for affinity labeling. Since solutions of BrAcT4 and of BrAcT3 undergo partial decomposition on evaporation to dryness, suitable procedures for the preparation of these hormones in solid form and for storage in solutions have been devised.     

Effect of toxic components of snake venoms on binding of substance P with rat brain membranes]

Ion-exchange HPLC is used for purification of the snake venom alpha-neurotoxins, chi-bungarotoxin, cytotoxins, and phospholipases A2. Among these purified polypeptides, phospholipases A2 are found to be the most potent in inhibiting the substance P binding to rat brain membranes, Ki approximately 10(-8) M. Other toxins are weak inhibitors (Ki greater than or equal to 10(-4)-10(-5) M), earlier data on the inhibiting activity of alpha-bungarotoxin being caused by the commercial preparations' contamination with phospholipase A2.     

Interference of a methotrexate derivative with urinary oncopterin [N2-(3-aminopropyl)biopterin] measurement by high-performance liquid chromatography with fluorimetric detection

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N²-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 μmollmol creatinine. The present results indicate that the peak found with elution from the C₁₈ column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

High-performance liquid chromatographic separation and partial characterization of human protamines 1, 2, and 3

A method for separating the three human protamines by HPLC of underivatized, total protamine extracts on a Nucleosil RP-C18 column is described. The identities of the three proteins have been confirmed by a combination of disc gel electrophoresis, amino acid composition, and primary sequence analysis. The results show that human protamine 3 elutes first, closely followed by protamine 2. Protamine 1 elutes later. The amino acid compositions and partial amino terminal sequences of human protamines 2 and 3 indicate that these two proteins are very closely related and suggest that they differ only by three amino-terminal amino acids.     

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the simultaneous determination of N²-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-5SA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.     

Routine and sensitive method for determination of nicorandil in human plasma developed for liquid chromatography with ultraviolet and m0ass spectrometric detection

A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid–liquid extractions, first with CH₃Cl and HClO₄ as denaturation reagent and second with addition of ethyl acetate and Na₂CO_3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 μm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC–MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Advances in sodium-ion coupled biogenic amine transporters.

The sodium-ion coupled transporters for 5-hydroxytryptamine (5HT), noradrenaline and dopamine function to reduce extracellular levels of biogenic amines. Over the past fifteen years selective inhibitors of these transport systems have been developed including fluoxetine, citalopram, paroxetine, litoxetine (for 5HT), nisoxetine, desipramine, maprotiline (for noradrenaline) and GBR-12935 (for dopamine). Some of these inhibitors, including drugs selective for noradrenaline transport and particularly those selective for the 5HT transport system are currently widely used in the clinical management of affective disorders. Selective biogenic amine uptake inhibitors have, in addition, provided tools to undertake molecular pharmacological and biochemical studies of their respective transporters. By this means, the rat brain 5HT and dopamine transporters have been identified as polypeptides with relative molecular masses of 73,000 and 80,000, respectively, using affinity-chromatographic purification and photoaffinity-labelling techniques. Recently, the biogenic amine transporters have been cloned and a comparison of their predicted amino acid sequences reveals that these proteins share a considerable degree of similarity with notably 12-13 transmembrane spanning domains. Perspectives for future fundamental and clinical research on biogenic amine transport systems using molecular biological techniques are discussed.     

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.     

Endogenous "ouabain-like" activity in bovine plasma

An endogenous inhibitor of the sodium pump has been detected and concentrated 1000-fold from bovine plasma. The steps of purification included deproteinization and extraction with methanol, removal of lipids by coextractions with a lipophilic solvent, desalting and further concentration by adsorption on C18-SepPack cartridges and HPLC fractionation on a weak anionic exchange column. The material isolated displaces 3H-ouabain from brain synaptosomes, inhibits red cell membrane Na,K-ATPase without inhibiting Mg-ATPase or Ca,Mg-ATPase. Deproteinization of plasma by boiling may lead to appearance of non-specific inhibitors. The procedures developed should now permit isolation of sufficient amount of material for further purification and structural characterization.