Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Preparative isolation of adult human liver metallothionein isoforms

Preparative human liver metallothionein (MT) isolation is described. MT was saturated with cadmium to follow MT purification spectrophotometrically instead of by metal content and to increase the stability of the protein. A concentrated, MT-rich fraction of the liver cytosol was prepared by selective organic solvent (acetone or acetone/methanol) fractionation. Conventional gel filtration and ion-exchange chromatographies resolved two MT isoforms, MT-I and MT-II. When needed, purification of MT from other low-molecular weight proteins was further increased by gel filtration chromatography at zero ionic strength, i.e., in distilled water. Reversed-phase high performance liquid chromatography of both MT isoforms resolved further peaks sharing MT properties not only from the MT-I but also from the MT-II ion-exchange isoform. The results show that it is feasible to perform a human liver MT isolation from an entire human liver with a reasonable laboratory capability.     

A comparison of 3 methods of lipoprotein (a) isolation from human plasma]

Three methods for purification of lipoprotein (a) [Lp(a)] from human plasma were compared. Method I: two-stage ultracentrifugation with subsequent gel-filtration of Lp(a) containing fractions (1.063-1.090 g/ml) on Sepharose CL-4B. Method II: ultracentrifugation followed by affinity chromatography of plasma fraction (1.063 g/ml) on anti-apoB sorbent. Method III: affinity chromatography of the whole plasma on anti-apo(a) sorbent. The Lp(a) yield of these methods is 35, 54 and 41%, respectively. The method III is preferable of these three because it permitted high purification of a large amount of Lp(a) by single-step chromatography.     

Preparative scale isolation of sphingosine

A high-yield method is described for the preparation of sphingenine from galactosylceramide. Two successive cleavage reactions are used, alkaline butanol to form galactosylsphingosine, then acidic acetonitrile to form the sphingenine. A column purification procedure utilizing silica gel is described. The method is superior to previously described methods in its rapidity and avoidance of isomerization and contamination by racemization and rearrangement products.     

Preparative isolation of serum a albumin

A simple method is suggested for the preparative isolation of native albumin in the thick (6 mm) block of the agar-agar gel on the veronal-medinal buffer solution (pH 8.6, ionic strength 0.1). The method is based on the analytical horizontal electrophoresis. 0.8-1 ml of 10% solution of proteins was introduced into each of two starting trenches. 5h after the beginning of distillation in the anode edge of the plate at a distance of 35 mm from the start two collector slits were cut out perpendiculary to the protein movement. Then they were filled with the NaCl physiological solution and protein portions were taken thrice each half an hour. The purity of albumins was checked immunologically. The method may be applied for obtaining other individual proteins.     

Isofocusing of antigenic small nuclear ribonucleoproteins. II. Preparative isolation

In a companion report (T.B. Okarma, W.S. Schrier, and R. Feinbaum, 1985, Anal. Biochem. 147, 27-37) the behavior of small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels was characterized. This communication extends those findings and describes a gentle procedure for the preparative isolation of snRNPs in native form from cultured murine L-5178y leukemia cells using sucrose density gradient centrifugation, preparative isofocusing, and gel filtration chromatography. Isofocusing in granulated gels separated intact uridylic acid (U)-snRNPs from tRNA and La RNPs. The U-snRNPs remained immunoprecipitable by lupus antisera throughout fractionation. The final product obtained in 2% yield contained primarily U1 and U2 snRNAs and lesser amounts of U3, U4, U5, and U6, along with the core U-snRNP polypeptides A-G. The core polypeptides displayed apparent pI's which ranged from 4.5 to 9.5 when analyzed by two-dimensional gel electrophoresis. Proteins B (28,000), D (16,000), and E (13,000) exhibited isoelectric variants. The Sm determinant proteins B' (28,000) and E (13,000) isofocused as basic peptides with apparent pI's of 9.5 and 8.5, respectively. The purity of the final fractions compared well with that of immunoprecipitates and the procedure reproducibly generated yields of native snRNPs sufficient for in vitro studies of their biological function.     

A comparison of methods for the isolation and fractionation of reticulocyte ribosomes

1. Polysomes, ribosomes and pH5 enzymes were isolated from rabbit reticulocytes by acidifying the post-mitochondrial supernatant to pH6.0 to precipitate all ribonucleoprotein particles and about half the pH5 enzymes; the precipitate was redissolved in buffer, pH7.6, and fractionated by zone centrifuging. 2. The isolation of polysome-rich and ribosome-rich fractions from the post-mitochondrial supernatant was also examined. 3. Studies of the stability of polysomes revealed that dissociation into sub-units occurred when both bound and free Mg(2+) was chelated by EDTA or when the pH was increased above pH8.8.     

A comparison of extraction methods for the isolation of phospholipids from biological sources

Four classical methods, as well as a method presented in this paper, were compared as to their efficiency in extracting phospholipids from animal tissue. After the extractions, total lipids were separated quantitatively by DEAE-Sephadex chromatography into their acidic and nonacidic fractions. The two fractions were then further analyzed by gradient saturation high-performance thin-layer chromatography (HPTLC) combined with scanning photodensitometry after coloration with copper acetate. Of the five methods compared, the present and Christiansen's methods based upon single-phase solvent systems proved to be more efficient than biphasic extraction procedures. The undesirable discriminatory effect exhibited by biphasic solvent systems toward acidic phospholipids which were partly retained in the aqueous phase was confirmed by statistical evaluation of the HPTLC results. Total chromogenic response of acidic phospholipids extracted using biphasic solvent systems was shown to be lower by 10-35% in comparison to the single-phase method of Christiansen. The suitability of the present method for studies involving phospholipid synthesis was confirmed by monitoring the elimination of water-soluble compounds from the single-phase extracts using a classical phospholipid precursor, 2-[3H]glycerol-3-phosphate. The labeled compound was eliminated (99.3-100%) from the single-phase postcentrifugation supernatant, followed by DEAE-Sephadex chromatography.     

Preparative isolation of Band 3, the predominant polypeptides of the human erythrocyte membrane

Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.     

Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.     

Preparative isolation of polyphosphoinositide fractions from ox brain

A simple preparative method for chromatographic isolation of pure fractions of di- and triphosphoinositides (1-phosphatidylinositol 4-phosphate and 1-phosphatidylinositol 4,5-bisphosphate) from ox brain is described. Polyphosphoinositide fractions have been obtained by ion-exchange chromatography of the lipid extract using gradient elution with 0-0.6 M ammonium acetate in chloroform/methanol/water (20:9:1) from a DEAE-cellulose column. Before chromatography, divalent metal ions were removed from the lipid extract by passing through a Dowex-50 (H+) column and lipids were converted to the sodium salt by neutralisation with sodium hydroxide in methanol solution. After chromatography, fractions of di- and triphosphoinositides were precipitated in methanol/water mixture (1:1) by evaporation in a vacuum to a final concentration of about 4 M ammonium acetate. Necessary salts of di- and triphosphoinositides were obtained by passing the ammonium salts of the lipids through Dowex-50 (H+) and neutralising with corresponding base in methanol solution. About 0.35 mmol of diphosphoinositide and 0.63 mmol of triphosphoinositide were obtained from 1 kg of wet ox brain tissue.     

Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells.

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Preparative study on the isolation of single sarcomeres from rabbit skeletal muscle.

Single sarcomeres were prepared from fresh rabbit myofibrils by digestion with a calcium-activated factor (CAF). The rabbit single sarcomere has functional properties quite similar to those of single sarcomeres obtained from chicken muscle by a usual method. Thus it was found that the single sarcomeres obtained by CAF digestion were useful as a muscle model, though they were not completely intact.     

The measurement of human endometrial prostaglandin production. A comparison of two in vitro methods

Two in vitro methods for measuring human endometrial prostaglandin production were compared. Endometrial samples from eight patients were incubated over eight hours by a perifusion and a superfusion technique. The collected fractions were assayed by radioimmunoassay for PGE2 and PGF2 alpha. There was no significant difference between the perifusion and superfusion methods for the pattern and amount of PGE2 and PGF2 alpha production with time. Significantly higher production levels of PGE2 and PGF2 alpha were found in secretory phase endometria than in proliferative phase endometria. Histological examination of the tissue specimens by light and electron microscopy showed that both methods caused gross tissue damage after eight hours experimentation. The superfusion method produced more morphological damage than the perifusion method. However, no tissue damage could be detected after one hour of incubation with either method. Over an eight hour period neither the perifusion nor the superfusion technique appears to be a good indicator of in vivo endometrial prostaglandin production. Either technique used for only one to two hours may better reflect the in vivo situation.     

Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A.

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).