Molecular Sieve High Performance Liquid Research Articles

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus.

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.

Rapid isolation of apolipoprotein E from human plasma very low density lipoproteins by molecular sieve high performance liquid chromatography

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.

Lipophilin (proteolipid apoprotein) of brain white matter. Purification and amino acid sequence studies of the four tryptophan fragments.

The chemical cleavage of lipophilin (proteolipid apoprotein) from bovine brain white matter with HBr/dimethyl sulfoxide at the tryptophan residues, under conditions adapted to this hydrophobic protein, releases four fragments with approximate molecular masses 14 kDa (Trp I), 6.8 kDa (Trp IV), 5.2 kDa (Trp III) and 2.1 kDa (Trp II). These fragments have been separated and purified by a combination of solvent distribution, molecular sieve chromatography (Bio-Gel P-150) and high-performance liquid chromatography for automated Edman degradation and combined gas-liquid chromatography/mass spectroscopy. The complete amino acid sequences of Trp II and III and large sequences of Trp I are reported in this communication. The amino acid sequence of Trp IV and the sequences of peptides releasable from lipophilin by proteolytic enzymes (trypsin, thermolysin, subtilisin, chymotrypsin) have been described in previous reports from this laboratory. Despite two small gaps in the complete primary structure of lipophilin from myelin of central nervous system, our sequence data suggest the arrangement of four long hydrophobic sequences (30-40 apolar amino acid residues) within the hydrophobic core of the myelin lipid bilayer, linked by three hydrophilic regions at the aqueous membrane interphase. These features lend lipophilin the properties of a polytopic membrane protein.