Partial purification of rat and human serum factors inhibiting the G1-S transition in rat hepatocytes

A low molecular weight compound, which inhibits the G1-S transition in rat hepatocytes, was purified from rat trypsin-treated serum by DEAE-cellulose chromatography and high-performance liquid chromatography on three different stationary phases. This procedure led to a 34500-fold purification with a 29% yield. Inactivation of the purified material by specific enzymes showed that the inhibitor is a glycopeptide containing a peptide moiety, N-acetylneuraminic acid and galactose residues. Amino acid analyses indicated the possible existence of a pentapeptide structure. The same purification procedure was applied to the corresponding human inhibitor. Inactivation by specific enzymes showed that it is also a glycopeptide.     

Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Anti-insulin-like (diabetogenic) peptides in pituitary extracts: role of oxytocin

A peptide has been extracted and characterized from whole bovine pituitaries that has anti-insulin-like activities when assayed in rat adipocytes. This peptide has been purified approximately 100,000-fold, is homogeneous by thin-layer chromatography in three separate solvent systems, and shows a single peak by reverse-phase high-performance liquid chromatography. By these chemical criteria, as well as biological activity criteria (14CO2 production from D-[U-14C]glucose and D-[U-14C]glucose incorporation into glycogen in rat adipocytes], the peptide is indistinguishable from oxytocin. It reacts with anti-oxytocin antibody, and has an amino acid composition indistinguishable from purified oxytocin. The relationship between this material and other previously described anti-insulin or diabetogenic peptides is discussed, but it was not possible to conclude that this peptide, which has been purified to homogeneity and constant specific activity, is related to these previously described factors.     

Identification of linoleic and oleic acids as endogenous Na+,K+-ATPase inhibitors from acute volume-expanded hog plasma

Na+,K+-ATPase inhibitors have been found to exist in acutely saline-infused hog plasma, which also inhibit the specific binding of ouabain to Na+,K+-ATPase and the binding of digoxin to specific anti-digoxin antibody. Two of these inhibitors were purified by a combination of Amberlite XAD-2 adsorption chromatography and 3 steps of high-performance liquid chromatography. Reverse phase, high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry identified these substances as linoleic (18:2) and oleic acids (18:1). A significant increase in the ouabain-displacing activity was observed in hog plasma during saline infusion. The maximal level reached was approximately 10 times higher than that of the preinfusion plasma sample. The two unsaturated fatty acids contributed to approximately 52% of the total ouabain-displacing activity after 120 min of saline infusion. The increased fatty acid levels in volume-expanded plasma are sufficient for an extensive inhibition of Na+,K+-ATPase activity. These results strongly suggest that free unsaturated fatty acids in plasma regulate extracellular fluid volume in a pathological volume-expanded condition through modulation of Na+,K+-ATPase activity.     

Three novel oligosaccharides with the sialyl-Lea structure in human milk: isolation by immunoaffinity chromatography

We have determined the structures of three novel oligosaccharides isolated from human milk using the monoclonal antibody MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography. From the results of 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, their structures were deduced to be (formula; see text) These oligosaccharides bound to MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but bound to another sialyl-Lea structure-directed monoclonal antibody, NS 19-9, only weakly.     

Soluble heparin-binding lectins from human brain: purification, specificity and relationship to an heparin-binding growth factor.

1. An heparin-binding lectin activity was detected in soluble extracts of human brain. Three polypeptide chains were purified by affinity chromatography on heparin-Sepharose. Their Mrs estimated by polyacrylamide gel electrophoresis were 13,000, 14,500 and 16,000. 2. Several glycosaminoglycans were potent inhibitors of their hemagglutination activity. 3. From the pool of purified lectins three peaks were separated by reversed-phase high-performance liquid chromatography. They were indistinguishable by activity criteria (hemagglutination, stimulation of endothelial cell growth), and immunological relationship was found between one of them and acidic fibroblast growth factor (aFGF).     

Purification and characterization of human liver sorbitol dehydrogenase

Sorbitol dehydrogenase from human liver was purified to homogeneity by affinity chromatography on immobilized triazine dyes, conventional cation-exchange chromatography, and high-performance liquid chromatography. The major form is a tetrameric, NAD-specific enzyme containing one zinc atom per subunit. Human liver sorbitol dehydrogenase oxidizes neither ethanol nor other primary alcohols. It catalyzes the oxidation of a secondary alcohol group of polyol substrates such as sorbitol, xylitol, or L-threitol. However, the substrate specificity of human liver sorbitol dehydrogenase is broader than that of the liver enzymes of other sources. The present report describes the stereospecific oxidation of (2R,3R)-2,3-butanediol, indicating a more general function of sorbitol dehydrogenase in the metabolism of secondary alcohols. Thus, the enzyme complements the substrate specificities covered by the three classes of human liver alcohol dehydrogenase.     

Isolation, characterization and N-terminal amino-acid sequence of rabbit transferrin

Rabbit serum transferrin has been isolated and purified by ion-exchange column and high-performance liquid chromatography. The N-terminal amino-acid sequence of 32 residues was determined by automatic Edman degradation in a liquid phase sequenator. Of the first twelve residues sequenced previously three identifications were corrected. Comparison with the known transferrin sequences shows 15 common amino-acid residues. Comparison to human serum transferrin revealed that 37% of amino-acid residues were exchanged. Cys9 and Cys19 which are supposed to be involved in disulphide bridges, are conserved.     

Purification of the T lymphocyte growth factor interleukin-2 from culture media of human peripheral blood leukocytes (buffy coats)

Interleukin 2 was purified 100 000-fold to apparent homogeneity from the supernatants of mitogen-stimulated human blood leukocytes. A sequence of three purification steps was used: affinity chromatography on the bound dye cibacron blue, gel filtration on Ultrogel AcA44, and reversed-phase high-performance liquid chromatography on hexyl phase. The resulting interleukin 2 had a specific activity of 2 X 10(6) U/mg protein, and was free of pyrogenicity in the rabbit test. The final purification product showed two bands in sodium dodecyl sulfate/polyacrylamide gels with apparent molecular masses of 15 kDa and 17 kDa respectively. Both bands were biologically active.     

Toxic substance from a natural bloom of Microcystis aeruginosa.

A toxic substance contained in the blue-green alga Microcystis aeruginosa was purified and partially characterized. Toxic algal cells were collected from a highly eutrophic lake in Japan, and the toxin was purified by homogenization, ultrafiltration, gel filtration, and ion-exchange chromatography. The final preparation gave a single peak on high-performance liquid chromatography. The toxicity was somewhat less than that reported for other toxins from this alga. The water extract of 6.7 mg (dry weight) of cells and 72 microgram of the purified protein was required to kill a mouse (1 mouse unit). The main amino acids of the toxin were glutamic acid, asparatic acid, alanine, glycine, arginine, and leucine. The molecular weight of the toxin was 2,950 as determined by high-performance liquid chromatography.     

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.     

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.     

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.     

Purification and some properties of membrane-associated phospholipase A2 of human spleen

Membrane-associated phospholipase A2 was purified to homogeneity from human spleen. The enzyme was solubilized from the particulate fraction by the addition of KBr, and purified by reverse-phase high-performance liquid chromatography. The estimated molecular weight of the enzyme was 14,000. The enzyme had a pH optimum around 9.5, required the presence of Ca2+ for its activity, and hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylcholine.     

A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat.

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).     

Quantitative analysis of monosialogangliosides by high-performance liquid chromatography of their perbenzoyl derivatives

A quantitative high-performance liquid chromatographic method for the analysis of monosialogangliosides as their perbenzoyl derivatives has been devised. Samples containing as little as 3 nmol were converted to their perbenzoyl derivatives by reaction with 0.1 ml of 10% benzoyl chloride in pyridine at 60 degrees C for 1 hr. The products were purified by silicic acid chromatography and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a 50 cm X 2.1 mm LiChrosphere SI 4000 column and a linear gradient of 7-23% dioxane in hexane in 18 min. Detection was at 230 nm. The detector response was found to be proportional to the amount of monosialoganglioside analyzed. As little as 50 pmol of injected material could be conveniently quantitated. The overall yield from derivatization and chromatography, as determined with radiolabeled GM1, was found to be 86%. To take advantage of the high sensitivity of the HPLC, a small-scale isolation method for gangliosides was devised. This method coupled with HPLC isotope dilution analysis was used to analyze the GM3 content of 1 ml of human plasma.     

Interferon-induced proteins. Purification and characterization of a 15,000-dalton protein from human and bovine cells induced by interferon

Human interferons induce a protein of 15,000 daltons in human and bovine cells. This protein is located in the cytoplasm in a soluble form and is induced by concentrations of interferon which induce the antiviral state. Messenger RNA prepared from interferon-treated human and bovine cells contains a mRNA which yields on translation in vitro a protein similar in size to the 15-kDa protein induced by interferon in vivo. The human protein has been purified to homogeneity from interferon-treated human cells by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. A comparison of the peptides generated by V8 protease from the human and bovine 15-kDa proteins reveals that the two proteins are similar but not identical.     

A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography

A simple procedure for purification of tetrodotoxin (TTX) derivatives by high-performance liquid chromatography is described. Chemically oxidized TTX, C₁₁-nortetrodotoxin (nor-TTX), was purified and collected by reverse-phase chromatography. The separation of nor-TTX from unreacted TTX was excellent and recovery of nor-TTX was more than 90%. The isolated nor-TTX was further coupled with lysine, and the coupled product was purified again by high-performance liquid chromatography on a cation-exchange column. The separation of all compounds required less than 15 min. The uv monitoring at 230 nm allowed the detection of TTX derivatives at the 2- to 3-ng level.     

Production of 5-formyluracil from thymine in an in vitro active oxygen-generating system

Thymine was placed in a model active oxygen-generating system containing ferrous sulfate, EDTA, and ascorbic acid. The oxidative products of thymine were separated by Sephadex LH-20 chromatography and a reversed phase high-performance liquid chromatography (HPLC) into at least five major components. One of them had a UV spectrum characteristic of 5-formyluracil and mass spectrometric analysis of this material also indicated this material to be 5-formyluracil.