Permeation High-performance Liquid Chromatography Research Articles

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3′,5′‐cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH4)2SO4 precipitation, DEAE‐cellulose chromatography, chromatography on 3′,5′‐cAMP‐agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH4)2SO4 pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca2+, Mg2+ and Mn2+, whereas NaF, PPi and Fe3+ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An MI of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3′,5′‐cAMP‐agarose a phosphodiesterase was resolved that produced 5′‐AMP as sole reaction product.

Von Willebrand factor antigen in urine

Human urine was analyzed using a sensitive enzyme linked immunosorbent assay (ELISA) for von Willebrand factor (VWF) antigen. Urine of healthy persons contained VWF immunoreactivity. In the urine of a patient with severe von Willebrand disease, the VWF antigen was not detectable before but after intravenous infusion of von Willebrand factor-Factor VIII (VWF-FVIII) concentrate. The VWF antigen in normal urine was analyzed by gel permeation high performance liquid chromatography (HPLC). Three immunoreactive components of Mr 350 KDa, 60 KDa, and 20 KDa, respectively, were observed. Chromatography on concanavalin A-Sepharose revealed heterogeneity of the major 60 KDa component since only part of the material had affinity for the matrix. The 350 KDa material displayed affinity for Con A-Sepharose but not the 20 KDa. Gel electrophoresis combined with immunoblotting of normal urine gave similar results to those obtained by HPLC analysis. Immunoreactive components with apparent molecular weights similar to the largest urinary antigens were also observed in normal plasma but they were, nevertheless, not identical to the urinary antigens.

Plasma lipoproteins inactivate platelet-activating factor (PAF).

Inactivation of platelet-activating factor (PAF) by plasma was demonstrated by comparing platelet aggregation induced by PAF solution, which had been preincubated with plasma, with that induced by the control PAF solution preincubated with buffer. Plasma inactivated PAF in a volume-dependent manner while preincubation of ADP or collagen with plasma did not affect the platelet aggregating activity of these agents. These facts imply that the effect of plasma is specific to PAF.The pretreatment of plasma with diiospropylfluorophosphate abolished the effect of plasma on PAF. Pretreatment of plasma with p-bromophenacyl bromide, an inhibitor of phospholipase A2, did not affect the action of plasma on PAF. Although phospholipase A2 has been shown to hydrolyze PAF in vitro, the evidence warrants independence of the inactivating process on this enzyme.Plasma PAF inactivating activity was found to be associated with total lipoproteins which had been separated by ultracentrifugation, while lipoprotein-deficient plasma was completely inactive. Further fractionation of plasma by gel permeation high-performance liquid chromatography disclosed that the activity is associated with low-density and high-density lipoproteins.

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.

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.

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.

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.

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.

Identification of Collagen Chains as a Function of Cyanogen Bromide Peptide Patterns Using Gel Permeation High-Performance Liquid Chromatography

A gel permeation high-performance liquid chromatography system utilizing commercially-available silica-based gels has been developed for evaluation of the cyanogen bromide cleavage products derived from collagen a chains. The high efficiency and precision of the system permits unequivocal identification of various chains by inspection of the peptide elution pattern following a single run requiring 40 minutes. The system is sufficiently sensitive to permit analyses to be performed with as little 1.0 µg of sample, although the columns utilized may accommodate samples as large as 1.0 mg making the system useful for preparative purposes as well.

Biochemical evidence for the presence of two vitamin D-dependent calcium-binding proteins in mouse kidney.

Mouse kidney, a vitamin D target organ, was investigated for the presence of vitamin D-dependent calcium-binding proteins (CaBP). Mouse kidney cytosol was fractionated by several biochemical methods including gel filtration chromatography, gel permeation high performance liquid chromatography, and chromatofocusing. Mouse kidney was found to possess two CaBPs which completely differed biochemically and exhibited no cross-immunoreactivity. One had a molecular weight of 25,000 and a pI of 5.9. The other, with a molecular weight of 10,000 and a pI of 4.9, was biochemically identical with mouse duodenal 10,000 CaBP. In addition, mouse renal and duodenal 10,000 CaBPs were immunologically identical. Moreover, the 10,000 CaBP was the predominant CaBP in mouse kidney since the latter contained about twice as much 10,000 CaBP as 25,000 CaBP (in mol/mg of renal cytosolic protein). In vitro incorporation of [3H]leucine into renal 10,000 CaBP demonstrated that it is synthesized in situ by mouse kidney. Renal 10,000 CaBP was already present during fetal life, and reached its adult level during the first week after birth. The vitamin D dependency of both mouse renal 10,000 and 25,000 CaBPs was assessed by their decrease in vitamin D-deficient mice and subsequent rise after 1,25-dihydroxyvitamin D3 injection. The concomitant presence of substantial amounts of two vitamin D-dependent CaBPs in mouse kidney is peculiar to this organ, which might consequently provide a unique model for studying the hormonal expressions of 1,25-dihydroxyvitamin D3.

Purification of radiolabeled and native polypeptides by gel permeation high-performance liquid chromatography

Some results and observations concerning the use of protein columns are presented. The combined use of four protein columns having different fractionation ranges together with a volatile triethylamine formate buffer allowed the sieving of various polypeptides according to their molecular weights over a range of 500 to 150,000. The addition of 4 or 6 m guanidine-HCl permitted the reduction of aggregation with no sacrifice in resolution or linearity. With that denaturant, rapid separation, and molecular weight determination in the range 500–90,000 is easily accomplished. Moreover, sample recoveries as determined with radiolabeled proteins always exceeded 70% while radioimmunoassay techniques can be directly applied to the column eluate. Applications to quick identification of natural fragments of a serine protease, tonin, analysis of maturation products of pro-opiomelanocortin in an in vitro pulse experiment and finally quantitation by radioimmunoassays of pituitary peptides and elution of their 125I-labeled derivatives are described.

High performance gel permeation chromatography of bovine thyrotropin (TSH): effect of column stability and mobile phase variation.

Because of previously observed variability in the elution patterns of radiolabeled bovine TSH (bTSH) and bTSH bioactivity (in a particular bTSH preparation) on gel permeation high performance liquid chromatography (GPC), studies were conducted to examine the effect of different conditions on the elution of this material. Continued use of a Waters' I-125 column at pH 7.2 demonstrated progressive retardation in the elution of bioactivity and radioactivity in this highly-purified bTSH preparation, with progressive but incomplete separation of these two functions. With decreasing mobile phase pH the elution positions of TSH bioactivity and radioactivity advanced to a coincident peak near to the void volume (pH 4). Addition to the mobile phase of the ion pair reagents tetrabutyl-ammonium phosphate (PIC-A) and pentane sulfonic acid (PIC B5) produced marked alterations in the elution positions of TSH radioactivity and bioactivity, with separation of these functions in the presence of the former reagent. These data indicate the present limitations of GPC for the purification of TSH.

Controlled proteolysis by subtilisin as a probe for cyanide-induced conformational changes in Pseudomonas cytochrome oxidase.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c-551:oxidoreductase; EC 1.9.3.2), digested with subtilisin in the absence and presence of KCN, produces discrete, high molecular weight fragments. The presence of KCN alters the rate of this fragmentation but does not change the nature of the fragments. When digested in the absence of KCN, the oxidase gives a major product (A) which is enzymatically active and has an apparent Mr = 58,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of KCN, the major product (B) has Mr = 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but with gel permeation high performance liquid chromatography it has an apparent Mr = 92,000. This implies that product B is dimeric, as is the parent enzyme which has Mr = 110,000 by high performance liquid chromatography. Absorption spectra of product B, isolated by gel filtration, show that it contains only the heme d1 moiety. The digestion time course indicates that the rate at which several minor products are formed is also dependent on the absence or presence of KCN. These observations suggest that the binding of KCN to the heme centers induces a conformational change in the enzyme so that the heme c-containing portion of the protein, which is at one end of the intact enzyme, can be removed without disrupting the integrity of the heme d1-containing portion.

Separation of membrane proteins of H. halobium by gel permeation high performance liquid chromatography.

High performance liquid chromatography (HPLC) separation of the SDS-solubilized membrane proteins of Halobacterium halobium S9 was investigated with a gel permeation column, TSK G3000 or 4000 SW. When sodium phosphate buffer (pH 7.0) containing 0.1% SDS was used for elution, both the elution volume and the elution profile of protein peaks were highly dependent on the buffer concentration. On increasing the buffer concentration, the elution volume for each increased and the resolution was also improved. Protein was recovered quantitatively from the column in the buffer concentration range between 0.01 to 0.2 M. In contrast to the phosphate buffer system, the elution profile of the membrane protein was almost independent of the salt concentration when Tris-HCl (pH 7.0) buffer containing 0.1% SDS and various amounts of NaCl was used for elution. The best separation conditions finally determined for the membrane proteins of H. halobium using SW columns were as follows : 0.2 M Tris-HCl (pH 7.0) containing 0.1 M NaCl and 0.1% SDS as an elution buffer system, at a flow rate of 0.6 ml per min. Under the above elution conditions, nine major protein peaks could be determined in the SDS-solubilized plasma membrane preparation of H. halobium S9 within 40 min when the absorbance was recorded at 280 nm. The resolution obtained is comparable to that of SDS-PAGE.

Analysis of serum iron by gel permeation high-performance liquid chromatography

A high-performance liquid chromatographic procedure for the determination of serum iron is reported. Serum iron extracted with methyl isobutyl ketone was converted to dibenzoylmethane chelate (molecular weight 725), and it was separated from excess dibenzoylmethane (molecular weight 224) by gel permeation chromatography. The chelate was determined by measuring ultraviolet absorption at 280 nm. Good reproducibility, recovery, and correlation with the conventional colorimetric method were observed.