Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography.
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Animals↗
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Detection of labile zinc-binding ligands in biological fluids by modified gel filtration chromatography.
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Animals↗
Characterization of the carcinoembryonic antigen activity associated with cyst fluids of mucinous ovarian cystadenocarcinoma.
Cyst fluids from mucinous cystadenocarcinoma of the ovary show significant concentrations of carcinoembryonic antigen (CEA) activity. This CEA activity was compared to several colon CEA standards with respect to size, concanavalin A binding, and immunological activity. CEA activity in unfractionated ovarian cyst fluid was indistinguishable from CEA standards by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cyst fluid CEA activity showed identity with various CEA standards by double immunodiffusion in agar gels and by radioimmune competition assay. Attempts to purify the cyst fluid CEA by perchloric acid extraction prior to gel filtration chromatography were unsuccessful. The yield of soluble CEA activity following perchloric acid extraction averaged 10%, and in one cyst fluid, qualitative changes were noted in the soluble antigenic activity. Ovarian cyst fluid CEA activity was bound by concanavalin A-Sepharose 4B and specifically eluted with competing monosaccharide. Lectin affinity chromatography and gel filtration chromatography over Sepharose 4B and Sephadex G-200 can be used to purify cyst fluid CEA from whole fluid or lyophilized and reconstituted samples.
Carcinoembryonic Antigen↗
Bovine dihydropteridine reductase: purification by affinity chromatography and comparison of enzymes from liver and adrenal medulla.
Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5'-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.
Adrenal Medulla↗
Cell-surface antigens from human breast tumor cells.
Biochemical and immunologic studies on breast cancer with the use of cells from a human ductal cell carcinoma, BOT-2, were initiated. Antigens were extracted from the cells by mild sonication and purified by gel filtration chromatography. Only one of the three peaks from gel filtration chromatography reacted with antiserum prepared against whole BOT-2 cells. Analysis by polyacrylamide gel electrophoresis of the BOT-2 cell extract revealed many protein bands, whereas analysis of the antibody-reactive peak after gel filtration chromatography revealed fewer protein bands. Immunologic tests to identify human serum antibodies against BOT-2 cells or cell extracts were performed by fixed cell immunofluorescence, living cell membrane immunofluorescence, and indirect hemagglutination. Depending on the test, the sera from women with diagnosed, untreated mammary cancer were positive in 45--80% of the cases, whereas the sera from women without apparent breast diseases (controls) were positive in only 5--10% of the cases. The results suggested that the antigens from the BOT-2 cells will be useful in understanding the processes involved in human mammary neoplasia.
Antibodies, Neoplasm↗
Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum.
The corticosteroid-binding globulin from guinea pig pregnancy serum was purified by the sequential use of affinity chromatography, hydroxylapatite chromatography, and gel filtration chromatography at a cumulative yield of 80%. The protein was found to be homogeneous by analytical gel electrophoresis, equilibrium sedimentation ultracentrifugation, immunoelectrophoresis, and stoichiometry (1:1) of steroid binding. Guinea pig corticosteroid-binding globulin has a molecular weight of 43 300 and contains 29% carbohydrate. The intrinsic fluorescence of the corticosteroid-binding globulin is quenched by about 73% when 1 mol of cortisol is bound. The association constants (pH 7.4) at 4 and 37 degrees C are 2.5 X 10(7) and 1.5 X 10(6) M-1 for cortisol and 1.4 X 10(6) and 0.2 X 10(6) M-1 for progesterone, respectively.
Amino Acids↗
Isolation and identification of a small molecular weight inhibitor of cyclic nucleotide phosphodiesterase from bovine brain.
In a search for endogenous regulators for cyclic nucleotide phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), we found that the ultrafiltrate of bovine brain homogenate contained a cyclic nucleotide phosphodiesterase inhibitor. The inhibitor-containing fraction was further purified by ion-exchange column chromatography and gel filtration chromatography. The purified inhibitor was found to be a small molecular weight compound which had a maximum absorption at 248 nm. This compound was identified by thin-layer chromatography and high-pressure liquid chromatography as hypoxanthine. We suggest that hypoxanthine may serve as an endogenous regulator for the hydrolysis of cyclic nucleotide by cyclic nucleotide phosphodiesterase.
3',5'-Cyclic-AMP Phosphodiesterases↗
Purification and chemical characterization of papain-solubilized histocompatibility-2 antigens from mouse liver.
A large scale purification of histocompatibility-2 (H-2) antigens from mouse liver is described. The antigens were solubilized by a limited papain digestion of a crude preparation of liver membranes (strain A/J) and purified by ion exchange chromatography, gel filtration, affinity chromatography, and isoelectric focusing. The overall degree of purification of H-2Kk was 1,300-fold and that of H-2Dd was 1,500-fold; approximately 8 mg of purified H-2a antigens were obtained from 1 kg of liver. The purification was followed by a sensitive radioimmunoassay in which H-2a-containing fractions were used to inhibit the binding of 125I-labeled H-2a to appropriate antisera. H-2Dd and H-2Kk co-purified through all the steps but the concentration of H-2Kk was 2- to 3-fold higher than that of H-2Dd in the liver homogenate as well as in the purified H-2 preparation. beta 2-microglobulin was initially present in a 3- to 10-fold excess over H-2 in the liver homogenate, but the purified H-2 preparation contained approximately 2 mol of alloantigenic heavy chain/mol of beta 2-microglobulin. Isoelectric focusing and disc-gel electrophoresis showed a charge heterogeneity of H-2, with a mean isoelectric point of pH 4.9. Electrophoresis on sodium dodecyl sulfate gels showed one band. Denaturing conditions were required to remove beta 2-microglobulin and small amounts of impurities from H-2. The amino acid sequence of the first 27 residues of the isolated heavy chains was determined.
Amino Acid Sequence↗
The extracellular metalloprotease of Serratia marcescens: I. Purification and characterization.
An extracellular protease of Serratia marcescens produced during growth on skim milk medium was isolated by ethanol precipitation. The protease was purified by salt fractionation, DEAE-cellulose ion exchange chromatography and gel filtration chromatography on Agarose P-100. It has a broad optimum from pH 6.0 to 9.0 and a temperature optimum of 45 degrees C for proteolytic activity on casein. It was classified as a metallo-protease by virtue of its inactivation by metal-ion chelators and reactivation by ferrous ions. Proteolytic activity was not affected by diiso-propylfluorophosphate, p-chloromercuribenzoate and dithiothreitol.
Binding Sites↗
Characterisation of the small molecular weight apolipoproteins from pig plasma very low density lipoprotein.
The small molecular weight apolipoproteins of pig very low density lipoprotein were investigated following their separation by gel filtration and ion exchange chromatography. Gel filtration through Sephadex G-200 in 6 M urea, produced essentially the same elution profile to that obtained after filtration of human very low density apolipoprotein. However, separation of the pig Sephadex fraction corresponding to human C proteins on DEAE-cellulose columns revealed the presence of only one major peptide and minor quantities of several others. Some properties of three apparent homogeneous fractions and one heterogeneous DEAE fraction were investigated. Unlike human apoprotein CII apoprotein, none of the pig peptides studied activated cow's milk lipase and sialic acid was not detected in any of the three purified C peptides of pig VLDL. The amino acid compositions of the pig peptides were different to those reported for human C apoproteins. The carboxy terminal residue of the major pig C peptide was shown to be serine. The differences so far revealed between pig and human C peptides need further investigation especially since this animal is regarded as a suitable model for investigating human lipoprotein metabolism and the development of atherosclerosis.
Amino Acid Sequence↗
A study of the single polypeptide nature of rhodanese. A comparison of different preparations.
The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.
Animals↗
Fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages.
The medium of cultured, SiO2-treated peritoneal macrophages contained a factor which enhances the incorporation of labelled proline to collagen and other proteins in granulation tissue slices, cells and polysomes. Simultaneously, the activity of alkaline RNase in the whole medium was decreased in comparison with the corresponding control. Polyvinylpyridine-N-oxide, PVNO, protected the macrophages against SiO2. Latex-particles and E. coli lipopolysaccharide decreased the RNase activity in the macrophage medium, but unlike SiO2 did not cause liberation of the collagen synthesis-stimulating factor. Fractionation of the medium by gel filtration chromatography showed the SiO2-pretreatment to have caused a very significant decrease in the aggregation state of RNase. The fraction from gel filtration chromatography that contained the SiO2-liberated factor stimulating collagen synthesis also contained the disaggregated RNase. There was no RNase-activity in the control sample. A homogenous protein (mol. wt. 14,300) was isolated with repeated gel filtrations from the medium of silica-treated macrophages. It increased the incorporation of 3H proline and 3H thymidine into cultured granuloma cells.
Animals↗
Identification and behavior of the precipitating BLV antibodies in sera of leukotic cattle.
Precipitating bovine leukemia virus antibodies isolated from bovine leukemic sera by ion-exchange chromatography, gel filtration and affinity chromatography were identified as immunoglobulins of the IgG1 subclass and IgA class. They proved to be different with regard to molecular size and electrical charge. Immunoglobulins IgG2 and IgM as well as low-molecular components ranging in the microglobulin regions (less than 4S) failed to precipitate BLV antigens. Individual bovine leukemic sera showed differing precipitating activities against p24 and gp69 antigens. Precipitating monospecific gp69 antibodies represented exclusively the IgG1 subclass. Some unidentified antigens in disrupted BLV preparations showed serological relationship to p24 and gp69, respectively.
Animals↗
Preparation of dipeptidyl aminopeptidase IV for polypeptide sequencing.
Dipeptidyl aminopeptidase IV is a dipeptidylpeptide hydrolase (EC 3.4.14) which hydrolyzes bond at the carboxyl group of proline releasing X-Pro dipeptides from the amino-terminus of polypeptides. The enzyme was purified 440-fold in 37% yield from swine kidney by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration and affinity chromatography with dipeptide-substituted Sepharose 4B. The enzyme released X-Pro from all X-Pro-beta-naphthylamides and polypeptides tested. The released dipeptides were not further degraded, and were readily identified in digests. The enzyme is suitable for use in the dipeptidyl aminopeptidase method for sequence analysis of polypeptides.
Amino Acid Sequence↗
Characterization of the collagen synthesized by endothelial cells in culture.
[14C]Proline and [14C]lysine were incorporated into collagen by cultures of endothelial cells derived from calf aortae. The isomer 3-hydroxy[14C]proline accounted for 10% of the total hydroxy[14C]proline in the collagen isolated from the medium. Approximately 81% of the hydroxy[14C]lysine isolated from the medium was glycosylated, and 91% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosylgalactose. Gel filtration chromatography or acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the initially synthesized peptide chain of [14C]collagen had a molecular weight of about 135,000; after pepsin digestion this was converted to 115,000. The ratio of hydroxy[14C]proline to total [14C]proline x 100 in the pesin-resistant fraction was 59. When examined by immunofluorescence microscopy, the endothelial cultures stained positively with antiserum to (Type IV) collagen from basement membrane of bovine anterior lens capsule. The data indicate that cultured endothelial cells derived from calf aortae synthesize collagen that resembles that of basement membrane collagen.
Aorta↗
Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: effects of dietary manipulation.
To study the metabolism of the abnormal plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency we performed five dietary experiments designed to perturb their distribution and composition. Four patients with the disease were given successive diets that differed in triglyceride, carbohydrate, or cholestrol content, and after each dietary period the lipoproteins were analyzed by combinations of preparative and analytical ultracentrifugation, gel filtration, chromatography, and disc gel electrophorsis. Lowering the intake of long chain, dietary triglyceride descreased the concentrations of the large very low density lipoproteins, the large and intermediate low density lipoproteins, and the small high density lipoproteins by as much ad 79 %, but either increased or did not change the concentrations of the small very low and low density lipoproteins. Re-adding long chain triglycerdine to the diet generally reversed these effects, but increasing the dietary cholesterol without lowering the dietary triglyceride only decreased the concentration of plasma cholesteryl ester. We conclude that the concentrations of the large very low and low sensity lipoproteins, the intermediate-sized low density lipoproteins, and the small high density lipoproteins are related to the absorption and subsequent transport of long chain dietary fatty acids. Since these lipoproteins are rich in unesterified cholesterol and lecithin, two polar lipids that form a substantial part of the surfaces of chylomicrons, components of chylomicron surfaces may accumulate in the patient's plasma following enzymic removal of chylomicron triglyceride and contribute to several of the abnormal lipoproteins.
Acyltransferases↗
Characterization of two species of Fc fragments obtained from rat IgG2a by prolonged papain digestion.
Prolonged papain digestion of rat IgG2a produced two molecular species of Fc fragments, termed Fc(I) and Fc(II). Studies by gel filtration chromatography and polyacrylamide gel electrophoresis in SDS/urea indicated that the two subunit polypeptide chains in each Fc preparation were associated by non-covalent bonds only. By analytical ultracentrifugation Fc(I) was found to have a m.w. of 47,100 and a sedimentation coefficient of 4.08S. Fc(II) had a m.w. of 39,800 and a sedimentation coefficient of 3.83S. The m.w. for the subunit chains of Fc(I) and Fc(II) were 25,300 and 20,300, respectively, as determined by analytical ultracentrifugation under dissociating conditions. Calculation of the frictional coefficient ratios indicated that both Fc fragments possessed compact globular structures. The difference in size between these two Fc fragments probably was due to a loss of some carboxy-terminal residues in Fc(II). Both Fc fragments possessed nearly identical amino-terminal amino acid sequences. Papain cleavage occurred primarily between residues 233/234 and 234/235. The carbohydrate compositions of the two species of Fc fragments were similar. It was concluded that under acid and reducing conditions papain cleavage of rat IgG2a occurred to the carboxy-terminal side of the hinge region. Prolonged papain digestion led to secondary attack in the carboxy-terminal end of the CGAMMA3 domain at an unidentified site, or sites, producing a stable second species of Fc fragments.
Amino Acids↗
[Study of leukotoxic activity of Cl. perfringens type A toxin and its fractions obtained by gel filtration and ion exchange chromatography].
In gel-filtration of Cl. perfringens type A toxin on Sephadex F-100 and F-50 there was revealed relationship between the leukotoxic activity and a high-molecular component. A method of ion-exchange chromatography on a column with DEAE-Sephadex A-25 from the Cl. perfringens toxin there were obtained 8 fractions 3 of which possessed a marked leukotoxic activity: the capacity to destroy neutrophils in the Svejcar and Vancurik test and to-depress the phagocytic activity of leukocytes. Lecithinase and lethal activity was revealed in one of these fractions only. Leukotoxic fractions differed by the capacity to destroy neutrophils and to decrease their phagocytic activity.
Cells, Cultured↗