Sephadex Gel Chromatography Research Articles

Hepatic clearance of gastrin and cholecystokinin peptides

The purpose of this study was to examine and quantify hepatic uptake and degradation of gastrin and cholecystokinin peptides. Rat livers were perfused in situ, without recirculation, with Krebs-Ringer bicarbonate (pH 7.4) containing 10% bovine erythrocytes, gassed with 95% O2-5% CO2. Gastrin and cholecystokinin peptide fragments of various lengths were injected into a portal vein via a sidearm syringe for over 1 min, and hepatic venous effluent was collected every minute for 20 min. After injection of 125I-labeled gastrin peptides more than eight amino acids in length, >95% radioactivity appeared in the hepatic venous effluent, all of which was intact peptide, as determined by immunoprecipitation, trichloroacetic acid precipitation, and Sephadex gel chromatography. Decreasing the chain length to eight or less amino acids resulted in progressive increases in hepatic uptake and degradation of gastrin fragments. Injected cholecystokinin peptides greater than seven amino acids in length traversed the liver intact, whereas smaller cholecystokinin peptides were cleared by the liver. The liver eliminated >90% of the biologically active carboxyl-terminal tetrapeptide amide common to gastrin and cholecystokinin. The results of this study indicate that gastrin and cholecystokinin peptides longer than seven amino acids traverse the liver without significant degradation, however, smaller peptides are progressively cleared during hepatic transit. Therefore, if small gastrin and cholecystokinin peptides from antral and intestinal mucosa are released and reach the portal venous circulation, hepatic inactivation would appear to prevent them from reaching the systemic circulation, precluding their significant contribution to gastric acid secretion and other physiologic functions.

Human Leukocyte Elastase-Like Proteinase Purified by Affinity Chromatography with Suc-L-Tyr-D-Leu-D-Val-pNA, and Its Identification with Human Spleen Fibrinolytic Proteinase

Elastase-like proteinase ( ELP ) extracted with 2 M NaClO4 from human leukocytes was purified by a new affinity chromatography technique with Suc-L-Tyr-D-Leu-D-Val-pNA, following delipidation, salting out and Sephadex gel chromatography. The purified preparation contained practically no chymotrypsin-like proteinase activity, and it was homogeneous on SDS polyacrylamide gel electrophoresis. The enzyme so purified readily degraded fibrin, fibrinogen, elastin and -Val type synthetic peptide substrates, such as Suc-L-Ala-L-Tyr-L-Leu-L-Val-pNA and Suc-L-Tyr-L-Leu-L-Val-pNA. A special increase in ELP activity by adding chaotropic ions was observed. The enzymatic properties of the ELP were very similar to those of spleen fibrinolytic proteinase (SFP). ELP and SFP were identified immunologically using mice antisera against purified ELP .

Carbohydrates in humic and fulvic acids from sediments of Suo sound, Japan

Vertical distributions of carbohydrates in humic and fulvic acids isolated from coastal sediments in Suo sound were investigated by gas chromatography and Sephadex gel chromatography. Humate carbohydrates were composed of fulvic acid carbohydrates (70 to 95%). Apparent molecular weight distribution of fulvic acid carbohydrates in vertical direction of sediments was 3·5 to 7·4% below molecular weight (M.W.) 1000, 32 to 42% for M.W. 1000 to 5000, 7·4 to 24% for M.W. 5000 to 10 000, 11·5 to 15·5% for M.W. 10 000 to 25 000, and 28 to 34% for M.W. over 25 000, respectively. Humic acid carbohydrates are present in high molecular weight over 25 000. Fulvic acid carbohydrates decreased with increasing depth. A decrease of humate carbohydrates occurred between the surface layers to 20 cm depth, below 20 cm vertical change of carbohydrates was not observed.

Colchicine stimulates the rate of contraction of heart cells in culture.

Microtubules have been demonstrated in intact heart muscle as well as in cultured myocytes. To better understand what role these filaments may be playing in the regulation of cardiac function we have used the microtubule disrupting agent colchicine and examined its effect upon the rate of beating of rat heart cells. Colchicine, but not the inactive stereoisomer lumicolchicine, increased the myocyte rate of spontaneous contraction in a dose dependent manner. This effect was clearly distinguishable from the positive chronotropic effect of isoprenaline and unlike isoprenaline was not blocked by propranolol. Colchicine was without effect on the in vitro activity of adenylate cyclase assayed in a myocyte homogenate. The binding of 3H-colchicine to cultured heart cells increased with a time course consistent with the increase in heart rate. Subcellular distribution and sephadex gel chromatography demonstrated that approximately 30% of the cell associated colchicine comigrated with tubulin. Measurements of total myocyte tubulin by 3H-colchicine binding indicated that tubulin represents about 0.04% of the total heart cell protein.

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage nonspecific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.

Partial Characterization of Eyestalk Hormones Controlling Molt and Gonadal Development in the Spiny Lobster Panulirus Argus

ABSTRACT The eyestalk neurosecretory system of Crustacea contains several peptide factors affecting various important physiological processes. Gonad inhibiting hormone (GIH) and a molt inhibiting hormone (MIH) have been separated and partially characterized using Sephadex gel chromatography and polyacrylamide gel electrophoresis. The eyestalks of the spiny lobster Panulirus argus were the source for these isolations, and activity was bioassayed in the fiddler crab Uca pugilator. Gonad inhibiting hormone has an apparent molecular weight near 5,000 daltons. Molt inhibiting hormone does not induce gonadal inhibition in fiddler crabs, but it does delay the onset of the molt cycle in eyestalkless crabs. Melanin dispersion in intact Uca pugilator was inversely correlated with gonadal growth.

Association of liposomes with the isolated perfused rabbit heart.

Liposomes were prepared from a mixture of either phosphatidylcholine (PC) and cholesterol (Ch) (7:2) (neutral), PC, dicetylphosphate and Ch (4:1:3) (negative), or PC, stearylamine and Ch (4:1:3) (positive). In addition, as the lipid phase-liposomal marker, 3H- or 14C-Ch was added. Alternatively, 14C-labelled mannitol, glucose or inulin was used as the aqueous phase-marker in some experiments. The liposomes were purified by Sephadex gel chromatography and by using microfilter. After 4 h of sonication, 95% of the total liposomes were found to be smaller than 1.2 microns. The entrapment volume was calculated to be 0.9-1.2 microliters/mumole of lipid. The ratio of lipid radioactivity and aqueous phase-radioactivity, which were found in a non-filtrable portion of the perfusate, remained constant during a heart-perfusion period of 30 min, indicating that the liposomes were stable during the experimental period. The wash-out experiment indicated that the liposomes were distributed in a space of the perfused heart nearly as large as the mannitol space. The liposomes were rapidly taken up by the heart during perfusion in a Langendorff manner. The positive liposomes were taken up by the perfused nonischemic heart at a greater rate than were the negative liposomes. The results with perfused ischemic hearts were equivocal. The liposomal label was found to be distributed unequally in the subcellular fractions, namely, relatively greater amounts (per mg protein) of liposome-bound radioactivity of Ch or mannitol were found in the microsomal fraction than in the mitochondrial or cytosolic fractions of the perfused heart. This distribution pattern was not influenced by the electrical charge of liposomes or by the oxygenation state of the heart perfusion. The accumulation of the liposomal label in the microsomal fraction found in the heart perfusion experiment could not be observed in the experiment in which tissue slices were incubated in the presence of liposomes, or in the experiment in which free mannitol was administered in the heart perfusion.

Heterogeneity of low molecular weight epidermal structural proteins of chick embryonic tarsometatarsal skin: Effect of hydrocortisone on its accumulation with reference to differentiation of epidermal cells

Cornified layers of newly hatched chick shank skin were solubilized in 8 M urea by reduction followed by S-carboxymethylations. On Sephadex gel chromatography the solubilized proteins were separated into two distinct protein fractions (protein A and protein B). SDS-gel electrophoresis showed that the smaller protein fraction (protein B) contained two molecules with molecular weights of 15000 and 17000. Protein B was resolved into several fractions, p I 4.5–5.2, by preparative isoelectric focusing in the presence of 6 M urea and 0.1% Nonidet P-40. The fractions all contained the two molecules ( M r 15000 and 17000). The amino acid compositions of these fractions and 2-dimensional electrophoresis of epidermal protein by the method of O'Farrel indicated that the two proteins are each heterogeneous with respect to charge for reasons of microheterogeneity in the amino acid composition and varying extent of phosphorylation. When chick embryonic skin was cultured in a chemically defined medium, hydrocortisone, which induces the synthesis of protein A and results in keratinization of epidermis (Sugimoto, M., Tajima, K., Kojima, A. and Endo, H. (1974) Dev. Biol. 39, 295–307), did not accelerate the accumulation of protein B in epidermis; the normal pattern of protein B was not formed in epidermis of cultured skin with or without the hormone. Actinomycin D did not inhibit the synthesis of protein B, suggesting that the mRNA for this protein has a long life.

Binding of 3H-Sertoli cell factor to rat anterior pituitary in vitro.

The binding characteristics of Sertoli Cell Factor (SCF) to the anterior pituitaries were examined using tissue homogenates and cultured cells. Tritium-labeled SCF (3H-SCF), synthesized by rat Sertoli cells in vitro in the presence of 3H-leucine and partly purified by Sephadex gel chromatography, was used to the ligand. The binding of 3H-SCF to the pituitary homogenates or cultured cells was considerably greater (3-6X) compared to homogenates of brain, kidney, liver, spleen and testis. The pituitary binding at 37 C occurred after approximately 60 time dependent, and directly related to the receptor concentration. Maximum binding at 37 C occurred after approximately 60 min of incubation. Over 90% of the bound 3H-SCF was competitively inhibited by an excess of unlabeled SCF, but not by other substances. There was a good correlation between the binding of 3H-SCF and its biological activity, as evidenced by a selective suppression of basal FSH release in pituitary cell cultures. These data indicate that, in analogy to other protein hormones, specific binding to target cell receptors may represent an initial step of SCF action in the pituitary. The binding should also prove valuable in the development of a rapid radio-receptor assay for SCF and possibly for other inhibin preparations.

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.

Chlorination Products of Humic Acid (Panel Discussion Mechanism of Trihalomethane Formation) (Proceedings of the 8th Symposium on Environmental Pollutants and Toxicology)

Reaction mechanism of humic acids of different origins with chlorine were studied. Volatile chlorinated substances produced by chlorination of humic acid were identified, and possible formation mechanism of one of these substances, chloropicrin, was investigated. Two kinds of humic acids showed different patterns of formation of chloroform and the other chlorinated substances under various conditions for chlorination. High performance liquid chromatography, Sephadex gel chromatography and infrared spectrophotometry of humic acid and chlorinated one demonstrated that these differences are derived from different functional groups in a molecule of humic acid, and that, in addition, humic acid is decomposed to lower molecular weight substances having molecular weight of about several thousands and higher chloroform formation potential. Volatile chlorinated substances were identified to be chloropicrin and carbon tetrachloride besides chloroform by GC-MS with a computerized data system. Precursors for the chloropicrin formation were amino acids such as alanine, threonine, ornithine, glutamic acid and glycine ; nitro- or nitrosophenols ; nitro- or nitrosoresorcinols ; nitromethane and nitroethane. Chloropicrin was produced by chlorination of humic acid and resorcinol in the presence of nitrite. As for mechanism of chloropicrin formation by chlorination of resorcinol in the presence of nitrite, it was recognized that nitroso-substitution of resorcinol occurred in the presence of nitrite and produces nitrosoresorcinol, which was oxidized to nitroresorcinol with hypochlorite, and finally nitroresorcinol formed chloropicrin by the method of the Rook's reaction of chloroform formation from resorcinol.

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl2 for activity. The following Km-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl2: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The Km of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 μM, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.

In vitro characterization of the mechanism of insulin degradation and the effect of chloroquine

The cultured rat hepatoma cell line, MH 1C 1, inactivates 125I-labeled insulin by a temperature dependent mechanism. The estimated K m for insulin degradation is 1.4 · 10 −7 M and the V is 2.5 · 10 −10 mol/10 6 cells/h. The iodocompounds released from cells preincubated at 0°C with 125I-labeled insulin are immunoreactive with anti-insulin antibody, while the iodocompounds released from the cells incubated at 37°C only reacts to a very small degree with anti-insulin antibody. The degradation products were analyzed by Sephadex gel chromatography. Sephadex G-75 gel filtration of iodocompounds derived from cells incubated at 37°C with 125I-labeled insulin for 5, 20 min and 1 h showed a progressive decrease in intact insulin, and an increase in the peak representing insulin breakdown products. Treatment with chloroquine, a lysosomal enzyme inhibitor, resulted in a large increase of cell-associated insulin compared to control cells. However, chromatographic studies of iodocompounds extracted from cells incubated with or without chloroquine show a similar pattern but differ in the size of the peak which represents the degradation products of 125I-labeled insulin. Furthermore, the iodocompounds released from the chloroquine treated cells were not immunoreactive with anti-insulin antibody. These results suggest that chloroquine inhibits the release of insulin degradation products from the cells.

Template-specific inhibitor for DNA polymerase isolated from cauliflower inflorescence

A novel inhibitory factor which greatly inhibits DNA polymerase activity was isolated from the apical portion of the cauliflower inflorescence during purification of DNA polymerases. It can be adsorbed on a DEAE-cellulose column, but not on CM-Sephadex or DNA-cellulose. The factor exclusively inhibits the incorporation of [ 3H]dTTP into DNA when poly(rA, dT 10) is used as the template primer, but not when activated DNA, heat-denatured DNA or native DNA is used as a template. The concentration of the factor in the reaction medium required for 50% inhibition is approx. 8 μg/ml. The factor is heatstable, is inactivated by trypsin, and has a maximum ultraviolet absorption at 278 nm. The molecular weight was estimated as 2500–3000 by Sephadex gel chromatography.

Isolation and characterization of multiple forms of rat liver lysozymes.

Multiple forms of lysozyme found in the rat liver were isolated and characterized from cellular organelles.Isolation of the enzymes was achieved by Sephadex gel filtration and chromatography on CM cellulose-column. The purity of the preparations was examined by electrophoresis on polyacrylamide gel. The nuclear lysozyme moved as a single band indicating homogeneity, whereas other subcellular lysozymes appeared heterogeneous due to presence of more than one band, thus showing partial purity. Although the subcellular lysozymes were similar with respect to enzymatic properties, pH, buffer molarity optima and electrophoretic mobility, differences were observed in elution patterns, responses to nuclear inhibitor and heat sensitivity. Nuclear lysozyme was distinctly different by these criteria as compared to other subcellular lysozymes.

Characterization of Pregastric Esterases

Sephadex gel chromatography and polyacrylamide gel electrophoresis were used to estimate molecular weights and demonstrate heterogeneity of pregastric esterases obtained from commercial extracts of sublingual tissue of calves, kids, and lambs. The molecular weights of calf, kid, and lamb esterases were approximately 172,000, 168,000, and 150,000. Each esterase preparation appeared to contain several enzymes that had similar molecular weights and electrophoretic mobilities.

Partial purification and characterization of natriuretic factor from rat kidney.

SummaryA partial purification of natriuretic factor from volume-expanded rat kidney has been achieved by Sephadex gel chromatography and acrylamide gel electrophoresis. This factor inhibits frog skin SCC and Na-K-ATPase from different sources in a dose-dependent manner. Activity is destroyed by incubation with trypsin or fresh kidney homogenate but not chymotrypsin.This work was supported by a group investigative award from the American Heart Association, Los Angeles affiliate. The authors are indebted to Ms. De-Anna Mackey and Ruby McCarty for secretarial assistance.

Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus.

Kanagawa phenomenon-associated hemolysin (K-hemolysin) was purified by Sephadex gel and ion-exchange column chromatography, after the culture supernatant had been adsorbed on and eluted from diethylaminoethyl-Sepharose CL-6B, and acid precipitated. K-hemolysin was a heat-stable and trypsin-susceptible protein with an apparent molecular weight of 44,000, the subunit of which was 22,000. The isoelectric point was 4.9. The minimum hemolytic dose was 0.1 mug/ml. The fifty percent lethal dose by intravenous injection was 1.4 mug. Electron microscopy of the small intestine of suckling mice orally challenged with the highest dose (50 mug) not only showed disappearance of epithelial cell microvilli, but also structural disturbances of the endoplasmic reticulum and mitochondrial swelling. One blueing dose representing permeability factor activity was 0.3 mug, and positive reaction in the rabbit ileal loop appeared at above 125 mug. Besides these data in experimental models, we discovered the appearance of an antibody in patients which neutralizes K-hemolysin during the course of the disease. This finding reinforces our view that K-hemolysin plays a most significant role in the pathogenesis of this enteric human disease.

Distribution and Biological Availability of Reactive High Molecular Weight Phosphorus in Natural Waters in New Zealand

Eutrophic lakes and many streams on the central volcanic plateau of the North Island, New Zealand, contain dissolved chemically reactive phosphorus which is not orthophosphate. Sephadex gel (G25-150) chromatograms reveal that one reactive component is of high molecular weight (MW > 5000) while a second component elutes in the same way as phosphate-phosphorus (PO4-P). The reactive high molecular weight phosphorus (RHMW-P) in streams generally forms only a small proportion of the dissolved reactive phosphorus (DRP). In some eutrophic lakes the proportion of RHMW-P in the DRP is high (70–80%). Each lake may have a characteristic distribution of the two forms of reactive phosphorus. In summer where DRP concentrations are low (<2 mg∙m−3) in lake surface waters, PO4-P seems to dominate over RHMW-P which is consistent with long 32PO4 turnover times which are found in many of the central volcanic plateau lakes. Algal growth responses to phosphorus uptake were similar for PO4-P and RHMW-P, but not all of the RHMW-P was taken up by Chlorella in bioassays. The proportion of the RHMW-P taken up by Chlorella was different for each lake examined, posing problems in relating DRP concentrations to algal growth responses in lakes.Key words: reactive phosphorus, phosphate-phosphorus, algae, Chlorella, growth, bioassay, freshwater analysis, molybdenum blue method, Sephadex gel chromatography

Concomitant production of beta-endorphin in ectopic ACTH/beta-LPH-producing tumors.

It is generally accepted that ectopic ACTH-producing tumors produce not only ACTH but beta-MSH or beta-LPH as well. Using a sensitive radioimmunoassay for beta h-endorphin, we have demonstrated the presence of beta-endorphin immunoreactivity with size heterogeneity according to Sephadex gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis in 6 ectopic ACTH/beta-LPH-producing tumors, providing further evidence for the role of a common precursor to ACTH and beta-LPH/beta-endorphin in the peptide biosynthesis in these tumors, in a manner similar to the biosynthetic events in the pituitary gland of several species.