Gel Permeation High-performance Liquid Research Articles

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-napthylamide (NA) bond of benzoyl(Bz)GlyLeuArgProGlyGlyLysArg2-NA which contains two likely processing sites, ArgPro and LysArg. On the basis of the ratio of V max to K m as a measure of substrate specificity, BzGlyLeuArgProGlyGlyLys Arg2-NA is about 50-fold better than BzGlyGlyLysArg2-NA. BzLeuArg2-NA and BzGlyLeuArgProGlyGly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (BzGlyGlyLysArg2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the M r is ≈160,000 but is ≈ 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, M r ≈ 70,000.

High-performance gel-permeation liquid chromatography of polypeptides on TSK-G2000-SW gels in dilute trifluoroacetic acid solutions

High-performance gel-permeation liquid chromatography of polypeptides on TSK-G2000-SW gels in dilute trifluoroacetic acid solutions

Characterization of a substance in rat serum which modulates prolactin release in vitro

A prolactin (PRL) inhibitory substance(s) present in rat serum has previously been shown to inhibit the release of PRL from rat pituitary cells in vitro (Hymer and Signorella, 1981). In this study, addition of rat serum to rat pituitary cells cultured in serum-free medium inhibited the release of PRL in dose-dependent manner. Neither the form of the released PRL molecule, nor its biological or immunological activity, was altered by rat serum treatment. Serum prepared from clotted blood previously incubated at 37°C always contained more PRL inhibitory activity than serum from blood incubated at 4°C; whole blood serum, but not plasma-derived serum, contained inhibitory activity. The inhibitor therefore comes from a formed element in the blood. Results of several experiments indicated that buffy coat cells, adherent blood cells and peritoneal cells released the inhibitor, but platelets and RBCs did not; it is suggested that a macrophage/monocyte is probably the cellular source of the PRL inhibitor. Gel permeation high-performance liquid chromatography (HPLC) of rat serum indicated that the inhibitory activity was associated with high molecular weight material, i.e. 115000–215000. The PRL inhibitory substance was (a) trypsin sensitive, (b) not extractable with butanol/diisopropyi ether, (c) precipitated at 40% saturated ammonium sulfate, (d) associated with material having an isoelectric point of 4.96 ± 0.26, and (e) not adsorbed with protein-A. These characteristics suggest the proteinaceous nature of the inhibitory substance. The inhibitor did not suppress PRL release until 9 h after addition to cultured cells, but decreased PRL synthesis was evident within 4 h of treatment. The inability of the dopanrine antagonist, haloperidol, to block the inhibitory effect of rat serum indicated that the material does not exert its effect through the dopamine receptor.

Hypnins, low-molecular weight peptidic agglutinins isolated from a marine red alga, Hypnea japonica

Four electrophoretically homogeneous agglutinins have been isolated from the aqueous ethanolic extract of the marine red alga Hypnea japonica by precipitation with organic solvents, gel filtration, and reversed-phase and gel permeation high-performance liquid chromatography. Since these agglutinins were found to be new peptides, they were designated hypnins A, B, C and D after the generic name of the seaweed. The major agglutinin, hypnin A, was a monomeric peptide with a molecular weight of 4200. It had an isoelectric point of 4.3, and contained large amounts of serine and glycine. The N- and C-terminal amino acids were identified as tyrosine and serine, respectively. On the other hand, hypnins B and C were suggested to be dimeric or trimeric forms of hypnin A or the related peptide, while hypnin D was quite different from the others. Hypnin A agglutinated not only animal erythrocytes other than human, but also mouse FM3A tumour cells. The hemagglutinating activity was inhibited only by glycoproteins with N-glycosidic sugar chains of a complex type. The hemagglutinating activity of hypnin A was not affected by heating for 30 min at 100°C or by addition of divalent cations. Hypnins B, C and D were similar to hypnin A in hemagglutination pattern and sugar-binding specificity.

Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung.

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.

Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.

Human Ile-Ser-bradykinin, identical with rat T-kinin, is a major permeability factor in ovarian carcinoma ascites.

Ascites from patients with metastatic ovarian carcinoma contains high amounts of an activity that increases vascular permeability, as easily detected by a rat skin test. Ascites was fractionated by gel permeation and reversed-phase high-performance liquid chromatographies. The fractions were analysed for permeability-increasing activity. In this way a peptide was isolated and identified as Ile-Ser-bradykinin by sequence and amino-acid analyses. It is identical with T-kinin which has previously been detected as a product of an acute-phase protein, T-kininogen, in rats but never in human material. The so far identified human kininogens, i.e. high- and low-molecular mass kininogens, can only release Met-Lys-bradykinin or its degradations products, as Ile-Ser-bradykinin is not a part of their structure. However, the present results provide evidence that the permeability factor Ile-Ser-bradykinin under certain conditions can be produced in considerable amounts also by human tissues.

Subcellular distribution of selenium in the liver from rats fed selenium from fish: selenocystine and inorganic selenite.

Four groups of rats of a normal selenium status were given different selenium compounds during a long-term feeding experiment (28 days). The selenium supplementations (per kg diet) were sodium selenite (1 mg), selenocystine (2 mg), and two different concentration levels of selenium from fish (0.1 and 1 mg). Differential pelleting of liver homogenates demonstrated that selenium was present in all the subcellular fractions, with a recovery of 55-60% in the cytosols. Gel permeation high-performance liquid chromatography of the cytosol fractions demonstrated the presence of protein-bound selenium at a molecular weight of 70,000 daltons. The subcellular distributions as well as the protein binding of selenium in the cytosols were identical in all dietary groups. This indicates a similar long-term liver metabolism of the four selenium compounds tested in the rat.

Isolation from fetal bovine serum of a peptide similar to the alpha chain of thrombin by reversed-phase high-performance liquid chromatography

During the preparation of erythrotropin from fetal bovine serum, a group of peptides co-eluted with this erythroid cell stimulating factor on semi-preparative reversed-phase high-performance liquid chromatography. They could be subsequently separated by a combination of reversed-phase high-performance liquid chromatography in the presence of heptafluorobutyric acid as ion-pairing reagent and gel permeation high-performance liquid chromatography. One of these peptides has been extensively purified. Partial amino acid sequence analysis indicated that fourteen of the seventeen N-terminal amino acids are identical with the N-terminal sequence of the alpha chain of bovine thrombin. The same isolation procedure could be useful for the identification of other major peptides of fetal bovine serum.

Immunofluorescence determination of IgG following high performance liquid gel permeation chromatography

The application of high performance liquid gel permeation chromatography (HPL-GPC) and immunofluorescence for the estimation of a protein was studied using human IgG as a model system following separation of the fluorescent immune complex from free fluorescent-labelled antibody on a column of HPL-GPC. Using FITC-labelled anti-human IgG Fab fragment and HPL-GPC, nanogram levels of IgG could be determined within 5 min of mixing the antigen and antiserum. HPL-GPC immunofluorescence analysis is sensitive specific, simple, rapid and highly versatile.

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium.

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

Phospholamban, a putative regulator of the Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum (SR), was purified from canine cardiac SR membranes. Cardiac SR was extracted with deoxycholate and fractionated with ammonium sulfate followed by gel permeation high performance liquid chromatography in the presence of the nonionic detergent, octa-ethylene glycol mono-n-dodecyl ether (C12E8), and KI. Further purification was achieved with CM-Sepharose CL 6B column chromatography in the presence of C12E8. The purified phospholamban showed a single band of 22,000 daltons on neutral sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412) and 27,000 daltons on alkaline SDS gels (Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685). Boiling of phospholamban in 2% SDS produced total conversion into the lower molecular weight component on SDS gels (11,000 on Laemmli gel and 10,500 on Weber and Osborn gel). The apparent molecular weight of phospholamban on SDS gels was slightly increased by cAMP-dependent phosphorylation. The extent of phosphorylation catalyzed by cAMP-dependent protein kinase in the purified phospholamban preparations was about 42 nmol of phosphate/mg of protein when the protein concentration was determined by the method of Lowry et al. (Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275), or 138 nmol/mg of protein based on the protein concentration estimated by the dye absorption method. Rabbit antisera were prepared against purified phospholamban. The obtained antisera were found to bind to purified phospholamban as well as that in cardiac SR. No reaction was detected in fast skeletal muscle SR by immunofluorescent staining of Western blots. The present preparation of purified phospholamban and the antisera should facilitate further understanding of the regulatory action of phospholamban on the calcium pump ATPase.

Gel permeation high performance liquid chromatographic fractionation of sawscaled viper ( [formula omitted][formula omitted]) venom: clotting and esterolytic activities : By R.C. SCHAEFFER, Jr., D. Marsh, S-M. Chilton, R.R. Renkiewicz and R.W. Carlson. Dept. Med., Wayne State Univ. Sch. Med., Detroit, MI 48201 USA

Gel permeation high performance liquid chromatographic fractionation of sawscaled viper ( [formula omitted][formula omitted]) venom: clotting and esterolytic activities : By R.C. SCHAEFFER, Jr., D. Marsh, S-M. Chilton, R.R. Renkiewicz and R.W. Carlson. Dept. Med., Wayne State Univ. Sch. Med., Detroit, MI 48201 USA

Lipid transport mechanism in the fish - II. Time course changes of lipid distribution in carp plasma lipoprotein after force-feeding with soybean oil.

Carp treated with the dorsal aorta cannulation was force-fed with 0.5ml of soybean oil per 100g body weight by a catheter. From the cannulated tube, 0.8ml of blood was collected at various intervals after feeding.Carp plasma separated into high and low molecular weight lipoproteins, namely Fraction A and B, based on the size of lipoprotein molecules using the high performance gel permeation liquid chromatography and the detection of triglyceride by enzymatic reaction. The molecular weight of Fraction A gradually increased up to 20h after feeding, then decreased as the time proceeded. Moreover, a higher molecular weight lipoprotein, Fraction A', appeared at 20h, followed by the disappearance during the next 48h. The time course changes of phospholipid and total cholesterol distribution in carp plasma lipoprotein fractions after feeding were similar to those of triglyceride pattern.It can be pointed out from these results that both of these two lipoproteins, namely Fraction A and A'. are primarily associated with the transport of lipids.

Purification of vitamin D binding proteins (group-specific components) in human plasma using a chromatofocusing method.

Human plasma vitamin D binding proteins (DBP, also denoted as group-specific components ; Gc) were highly purified from commercial human α-globulin (also denoted as Cohn Fraction IV ; HαG). Chromatofocusing separated the partially purified HαG into three DBPs which had the same molecular size in gel permeation high-performance liquid chromatography. They each exhibited binding affinity with 25-hydroxyvitamin D3 [25 (OH) D3] and cross-immunoreactivity against antihuman plasma group-specific components (Gc) antiserum. However, the three DBPs were completely different in charge on chromatofocusing, and gave distinct peaks having apparent isoelectric points of 4.64, 4.59 and 4.54. It is well known that the Gc has three phenotypes separable only by isoelectric focusing. The relationship between the three purified DBPs and the three phenotypes of Gc has not been clarified. However, we suggest that the chromatofocusing method may provide a convenient procedure for the purification of DBP from human plasma and commercial HαG.

Resolution of proteins in the kidney stone matrix using high-performance liquid chromatography.

The organic matrix accounts for 2-3% of the total stone weight and has been considered to play a role in stone formation and growth. Thus far, fractionation of the matrix proteins has been insufficient due to low resolution and reproducibility. In this report the matrix proteins of 22 stones were resolved by means of high-performance liquid chromatography. Following pulverization, the organic matrix was obtained by dialysis against EDTA. The average content of nondialyzable extractable proteins was 1.6% of the total stone weight. Analysis of the matrix proteins with high-performance gel permeation liquid chromatography and high-performance ion-exchange liquid chromatography has indicated that the protein composition of the stone matrix is identical regardless of the mineral composition. The major component of the matrix proteins was identified as glycoprotein and/or proteoglycan from their absorption to a concanavalin A Sepharose column. Higher molecular weight matrix proteins seem to be polymers or condensation products, since they have been degraded into lower molecular weight subfractions by sodium dodecyl sulfate treatment.

Chromatographic studies on the radiolysis of DNA in aqueous solution.

Radiation-induced degradation of double-stranded DNA from calf thymus in aqueous solution with sodium phosphate was studied by conventional gel chromatography and by high-performance liquid-gel permeation chromatography. Comparison of the data after radiolysis of aqueous solutions of DNA under anaerobic and aerobic conditions indicates that double-strand breakage is not enhanced by oxygen. An increase of ionic strength impedes the break-down of the DNA molecules, so that loss of DNA can only be observed at doses above 100 Gy. Only reactions of OH-radicals contribute to the fragmentation of DNA, while the presence of hydrated electrons, H.-or formate radicals does not lead to a loss of highly polymerized DNA up to doses of 1500 Gy. High-performance liquid-chromatography proved to be an excellent method of studying the degradation of macromolecules as a function of dose.

Application of a high-performance gel permeation liquid chromatographic procedure to the determination of binding of prednisolone to high-affinity binding sites in human serum

Application of a high-performance gel permeation liquid chromatographic procedure to the determination of binding of prednisolone to high-affinity binding sites in human serum

Presence of cystine-containing antifreeze proteins in the spruce bud worm, Choristoneura fumiferana

Using a trichloroacetic acid precipitation procedure and gel permeation high performance liquid chromatography, we have demonstrated the presence of an antifreeze protein in the second instar larvae of the spruce budworm Choristoneura fumiferana. This protein contained a significant amount of half-cystine and only a modest content of alanine. In a radioimmunoassay, it competed with antisera against a cystine-containing antifreeze protein from sea raven, Hemitripterus americanus, thus indicating their immunological cross-reactivity and possibly, some structural homology.

Isolation of two biologically active peptides, erythrotropin I and erythrotropin II from fetal calf intestine

A bioassay based on the measurement of thymidine incorporation into trichloroacetic acid-insoluble materials in erythroid cell suspensions from fetal calf liver was used as the assay for purification of two small peptides (erythrotropins I and II) from fetal calf intestine. The peptides were purified using reversed-phase and gel permeation high performance liquid chromatography (HPLC). The two peptides have very similar amino acid compositions and a molecular weight of about 3500 daltons. Erythrotropin II stimulated thymidine incorporation and potentiated the action of erythropoietin in cultures of erythroid cells from fetal rat liver.