Isolation and structure of somatostatin from porcine hypothalami.

The isolation and structure of somatostatin (GH-RIH) from pig hypothalami are described. This hormone was purified by preparative gel filtration, solvent extraction, countercurrent distribution in two solvent systems, ion-exchange and partition chromatography, and analytical gel filtration. The somatostatin activity was followed by in vitro bioassays and a radioimmunoassay. The isolated product was homogeneous chromatographically and had biological and immunological properties similar to synthetic somatostatin corresponding to the ovine hormone. The primary structure of porcine somatostatin was shown to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Other immunologically and biologically active form(s) of somatostatin were also detected.     

Somatostatin 28(1-12) in a somatostatin-secreting human medullary thyroid carcinoma cell line

We have identified a system, the TT human medullary thyroid carcinoma cell line, which we found to contain 31.3 +/- 27.7 ng of somatostatin 28(1-12) immunoreactivity/mg protein. Radioimmunoassay of gel filtration fractions showed that the major form of immunoreactive somatostatin 28(1-12) had a molecular weight of 1,500 daltons. During reversed-phase high pressure liquid chromatography, this 1,500-dalton species coeluted with synthetic somatostatin 28(1-12). Somatostatin 28(1-12) containing forms larger than 7,000 daltons were also observed. Further studies will be required to elucidate the route of processing of prosomatostatin. The fact that the products of prosomatostatin processing in these cells are similar to those in normal tissues indicates that the TT medullary thyroid carcinoma cell line constitutes a useful model for human somatostatin gene expression.     

Purification and characterization of human bronchial proteinase inhibitor

We describe a purification procedure for the human bronchial proteinase inhibitor which involves trichloroacetic acid precipitation of sputum followed by ion-exchange and gel filtration chromatography. The inhibitor shows a major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but exhibits microheterogeneity on high-resolution chromatography. It has a molecular mass of 15.5-16 kDa as determined by electrophoresis and gel filtration and is 90% active against leukocyte elastase. The amino acid sequence of the N-terminal portion of the inhibitor was determined and was found to be identical (through 29 amino acids) to that recently reported for the human seminal plasma proteinase inhibitor I (Seemuller et al. (1986) FEBS Lett. 199, 43-48).     

Purification of human alpha-2-macroglobulin by chromatography on Cibacron Blue Sepharose.

A simple and efficient procedure has been devised for the isolation of α-2-macroglobulin from human plasma (type 1-1 haptoglobulin). The primary step is gel filtration and affinity chromatography on Cibacron Blue Sepharose, which selectively removes albumin and retards lipoproteins and γ-globulin, while effecting the molecular sieving of the remainder of the plasma proteins. This results in the separation of about 40% of the α-2-macroglobulin as a homogeneous component. A second step, gel filtration on Ultrogel AcA 22, may be utilized to separate α-2-macroglobulin in contaminated fractions obtained after Cibacron Blue Sepharose chromatography.     

Use of electrophoresis and gel filtration to characterize human pituitary somatotropin preparations after storage.

Fresh and stored preparations of human pituitary somatotropin examined by different polyacrylamide gel electrophoresis techniques and by gel exclusion chromatography (Ultrogel) clearly demonstrated that the hormone undergoes a structural alteration during storage for 3 years. The formed aggregates were readily quantitated from the gel filtration profile and from polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). According to gel filtration the amount of aggregates in fresh and stored hormone was determined to be 2 and 10%, respectively, while SDS-PAGE indicated 6 and 14%.     

Rapid purification of tissue inhibitor of metalloproteinases from human plasma and identification as a gamma-serum protein.

A rapid method is described for the purification of human tissue inhibitor of metalloproteinases (TIMP) from plasma which involves immuno-affinity chromatography and gel filtration. The purified plasma inhibitor is immunologically identical with the TIMP previously purified from human amniotic fluid, human synovial fluid and human fibroblast culture medium. It is proposed that this inhibitor is identical with the plasma inhibitor previously named 'B1 anticollagenase', although the plasma inhibitor was shown to migrate as a gamma-serum component.     

Purification of prostatic acid phosphatase from human seminal plasma

Prostatic acid phosphatase has been isolated from human seminal plasma. The purification method utilizes gel filtration on Sephadex G100, ammonium sulfate precipitation and a series of chromatographical steps including concanavalin A Sepharose 4B, anion exchange and gel filtration chromatography. The final product appears homogenous when analyzed by gel filtration on Sephadex G100. It gives one major band on SDS polyacrylamide gels. The specific activity is similar to that obtained by other purification schemes. The yield of the method described above has allowed to set up a sensitive radioimmunoassay of prostatic acid phosphatase.     

On the accuracy of radioimmunological determination of somatostatin in plasma

We performed the following experiments to evaluate the accuracy of our newly developed radioimmunoassay for somatostatin: (1) Recovery of synthetic somatostatin added to human, porcine, and canine plasma with or without extraction with 67% acetone or 76% ethanol, using 3 different region-specific antibodies and, where applicable, 125I-labelled Tyr-1- or Tyr-11-substituted somatostatin or 125I-N-Tyr-somatostatin as tracers. The recovery of somatostatin corrected for losses inherent in the extraction procedure was close to 100%, and independent of species, antibody and tracer. Somatostatin 1-28 was extracted slightly less efficiently. Unextracted plasma interfered massively in the assay. (2) Pharmacokinetic experiments with infusion of somatostatin into 14 pigs and determination of metabolic clearance rate (MCR) and T-1/2. MCR was 27-38 ml/kg per min, independent of infusion rate (6.1 or 13 pmol/kg per min), extraction procedure or tracer. T-1/2 was 1.9 min. The infused somatostatin was not measurable in unextracted plasma. (3) Characterization of endogenous and exogenous, labelled and unlabelled somatostatin 1-14 in human plasma, using Sephadex G-50 columns at pH 7.5 and 9.0. Human plasma showed excess immunoreactivity eluting at the void volume whereas synthetic somatostatin was recovered quantitatively at the position of marker somatostatin when added to the plasma. The immunoreactivity of the tracers was decreased (125I-Tyr-11-somatostatin) or abolished (125I-N-Tyr- or 125I-Tyr-1-somatostatin) after incubation with plasma or void volume fractions of plasma subjected to gel filtration. Extracted plasma did not contain void volume immunoreactivity, but like whole plasma, small amounts of components which coeluted with intact somatostatin.     

Pancreatic somatostatin-like immunoreactivity suppresses gastric acid secretion in rats.

Somatostatin-like immunoreactivity (SLI) was extracted from the canine pancreas and purified by ion exchange, affinity chromatography and gel filtration. The 1600 dalton fraction, which is physicochemically similar to synthetic somatostatin was infused into the peripheral circulation of anesthetized rats and its effect upon gastric acid secretion was compared with that of synthetic somatostatin. Both synthetic somatostatin and pancreatic SLI in a dose of 7–8 μg/kg/h suppressed pentagastrin-stimulated gastric acid secretion. It is concluded that the highly purified 1600 dalton fraction of canine pancreatic SLI, like synthetic somatostatin, can exert biological activity upon the stomach of rats.     

Cultured human cells release soluble γ-glutamyltransferase complexes corresponding to the plasma b-GGT

Serum γ-glutamyltransferase (GGT) is thought to derive from the liver, but its values predict morbidity and mortality for several diseases, such as cardiac infarction, stroke, diabetes, renal failure and cancer. We assessed total GGT and its fractions in the culture supernatants of human cell lines (melanoma, prostate cancer, bronchial epithelium) by gel filtration chromatography. We also compared the GGT elution profile in plasma and the corresponding very-low-density lipoprotein (VLDL) fraction. All the cell lines tested released soluble GGT whose activity increased in parallel with the cell growth. Released GGT presented a molecular weight of 2000?kDa, identical to the b-GGT fraction of human plasma and corresponding to that of VLDL. But ultracentrifugation studies showed that b-GGT had a higher density than VLDL. The b-GGT present in human plasma can be produced by tissues other than the liver, thus explaining the increase of serum GGT observed in diseases of other organs.     

Depolarizing influences regulate somatostatin synthesis and processing in cultured cerebral cortical cells

There is increasing evidence that persistent depolarization plays a critical role not only in excitation-secretion coupling, but also in the mechanisms linking excitation of neuronal cells to long-term adaptative changes in biosynthesis of neuropeptides. Somatostatin (SRIF) release and synthesis are affected by numerous agents, such as high concentrations of potassium that cause depolarization of cellular membrane. In the present work, we tried to determine whether prolonged exposure to veratridine (VTD) regulates SRIF synthesis. We found that exposure to VTD (100 microM) resulted in the stimulation of total (cell content + media) immunoreactive SRIF (IR-SRIF). This effect was calcium- and sodium-dependent, since it was prevented when verapamil (VPM) 20 microM or tetrodotoxin (TTX) 1 microM were added simultaneously with VTD. Cerebral cortical cells were exposed to high potassium concentrations, and the nature of the IR-SRIF was characterized by high-pressure liquid chromatography (HPLC) or gel filtration. It was evident that chronic exposure to high potassium concentrations modified the elution profile of medium IR-SRIF on HPLC and gel filtration, causing an increase in somatostatin-28 (S-28) and a decrease in somatostatin-14 (S-14). The results indicate that chronic exposure to VTD or high potassium concentration increases immunoreactive somatostatin and augments synthesis of its high-molecular-weight forms. This suggests that chronic membrane depolarization activating sodium and calcium channels initiates the entry of calcium ions, which triggers somatostatin release and causes a depletion of its intracellular stores. The stimulation of somatostatin secretion could be coupled to synthesis of the peptide.     

Chemical identity of tryptensin with angiotensin.

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.     

Cloning,Overexpression, Purification and Preliminary Characterization of Human Septin 8

Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T _m of 54 °C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.     

Interference of a human leukocyte interferon preparation with stimulatory activities in leukocyte-conditioned medium for human hematopoietic stem cells

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of multilineage hemopoietic progenitors derived from human bone marrow. However, PHA-LCM prepared in the presence of a human leukocyte interferon preparation does not support mixed colony formation. Crude PHA-LCM preparations were characterized by gel filtration, affinity chromatography, and gel electrophoresis. The elution profile on Sephacryl S-300 of PHA-LCM prepared without interferon showed a distinct peak that stimulated the growth of pluripotent stem cells (CFU-gemm) and committed precursors (CFU-c, BFU-e). Gel filtration of PHA-LCM, prepared with 1000 U/ml of interferon, revealed a change in the elution profile. The eluted material demonstrated no growth-promoting activities. We conclude that the abolished stimulatory activity of PHA-LCM, prepared with human leukocyte interferon, might be due to a reduced production of stimulatory molecules, suggesting that interferon interferes with the molecular events required for colony formation of committed and noncommitted hemopoietic progenitors.     

A novel thrombin enhancement factor in human plasma

A protein has been isolated from human plasma by gel filtration followed by affinity chromatography with a derivative of wheat germ agglutinin and ion exchange chromatography. This protein showed one peak in high performance liquid chromatography but in gel electrophoresis, in the presence of sodium dodecyl sulfate and β-mercaptoethanol, revealed two major components of 74 kDa and 55 kDa. These results indicate that the protein probably exists as a complex of the two polypeptides. This protein complex enhanced platelet aggregation by thrombin while aggregation induced by ADP was not significantly affected. Similarly, the rate of thrombin action on fibrinogen and N-benzoylarginine ethyl ester as measured in a spectrophotometer was increased in the presence of this plasma protein. These results suggest the presence of a protein complex in human plasma which can directly interact with thrombin and enhance its reactivity.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Isolation and structure determination of two peptides occurring in human seminal plasma

Two previously unknown peptides with a high amount of polar amino acids were isolated from human seminal plasma by a combination of dialysis, gel filtration, ion-exchange chromatography, and RP-HPLC. Their structures were determined by gas-phase sequencing simultaneously considering the different peak intensities.     

Plasma vitellogenin in landlocked Atlantic salmon (Salmo salar Ouananiche): isolation, homologous radioimmunoassay and immunological cross-reactivity with vitellogenin from other teleosts

Vitellogenin was isolated by affinity chromatography and gel filtration from landlocked Atlantic salmon plasma. Vitellogenin was labelled with iodine-131 using iodogen and an homologous radioimmunoassay was developed. There was poor immunological cross-reactivity with vitellogenin or plasma from other teleosts. Parallelism of the vitellogenin standard to the displacement by plasma of vitellogenic salmon allowed the assay to be used to evaluate the seasonal concentration profile of vitellogenin in female adult salmon. Extracts of liver or ovary from female Atlantic salmon also yielded displacements parallel to the vitellogenin standard in the assay.     

Multiple forms of immunoreactive dynorphin in human pituitary and pheochromocytoma

Multiple forms of immunoreactive dynorphin (I-Dy) in human pituitary and pheochromocytoma were examined utilizing gel filtration and high performance liquid chromatography (HPLC). Gel filtration of I-Dy from these tissues revealed the major component in the position of Dy(1-17) and other minor components with large molecular weight forms. HPLC profile of this major component from gel filtration showed a large peak corresponding to the position of Dy(1-17) and small peaks corresponding to the positions of Dy (1-13), (1-12) and other unknown peptides. These results strongly suggest the presence of Dy(1-17) as the major component, and Dy (1-13), (1-12) or other unknown peptides as the minor components in these human tissues.