Purification of murine thymus leukemia antigen (TL). A quantitative assessment of limited proteolysis.

The murine thymus leukemia antigen (TL) has been solubilized from the tumor ASL1 and from an established cell line ASL1W, by papain digestion. When a 15-min digest was chromatographed on Sephadex G-200, two peaks of TL activity were eluted with apparent molecular weights of approximately 58,000 and 31,000. Chromatography of a 30-min digest under the same conditions resulted in elution of a single peak of activity with an apparent molecular weight of 58,000. Additional purification was carried out on the 58,000 molecular weight material by absorption to, and elution from DEAE-cellulose. The combination of gel filtration and ion exchange chromatography resulted in approximately a 150-fold purification.     

Antigens in immune complexes from patients with breast cancer

Summary Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (¹²⁵Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). ¹²⁵Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.     

Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Properties of fractionated chondroitin sulphate from ox nasal septa

1. Chondroitin sulphate was isolated from bovine nasal septa by precipitation with cetylpyridinium chloride after digestion of the tissue with papain. 2. The material was divided into two portions, one of which was partially degraded with testicular hyaluronidase. 3. Untreated and hyaluronidase-digested material were fractionated into a total of eleven subfractions by gel chromatography on Sephadex G-200 and Sephadex G-100 respectively. 4. Chemical analyses indicated that the composition of all the fractions was similar to that of chondroitin sulphate. However, electrophoresis revealed a charge-inhomogeneity in the low-molecular-weight fractions obtained after hyaluronidase digestion. 5. The physicochemical properties of the subfractions were investigated by sedimentation-velocity, diffusion and sedimentation-equilibrium studies, osmometry, viscometry and gel chromatography. The individual fractions were essentially monodisperse and showed molecular weights ranging from 2400 to 36000. 6. The relationship between the intrinsic viscosity and the molecular weight was [eta]=5.0x10(-6)xM(1.14), indicating that the chondroitin sulphate molecules assume a shape intermediate between that of a random coil and a stiff rod. 7. The relationship between the sedimentation constant and the molecular weight (>10(4)) was s(0) (20,w)=2.3x10(-2)xM(0.44).     

Purification and partial characterization of chick intestinal sucrase

The sucrase was purified from the small intestinal mucosa of the adult chick. Purification procedure involved solubilization with papain, ethanol precipitation, chromatography on Sephadex G-200 and DEAE-Sephadex. Several characters of the chick intestinal sucrase resembled those of the intestinal sucrase-isomaltase complex of some mammals (rabbit, rat and human).     

The N-terminal part of recombinant human tear lipocalin/von Ebner's gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs.

Human Tear Lipocalin/von Ebner's gland protein (TL) is a member of the lipocalin superfamily. The protein is secreted by a number of serous glands and tissues and is overproduced under conditions of stress, infection and inflammation. In addition to its typical affinity for lipophilic ligands it was recently found to be able to inhibit cysteine proteinases [van't Hof et al., J. Biol. Chem. 272 (1997), 1837-1841], probably due to the presence of amino acid motifs resembling the papain binding domains of family 2 cystatins. In this work we have used a recombinant protein to confirm the results obtained with native TL. The inhibitory activity of the recombinant protein against papain was dependent on the ratio of papain and TL. At higher papain concentrations, the N-terminal sequence of TL was cleaved off by the protease, indicating that it can act in an inhibitor- or a substrate-like mode. This behaviour resembles that observed with certain chicken cystatin mutants. Using a recombinant TL mutant we found that the two Leu residues (Leu4-Leu5) contained within the first cystatin-like motif are absolutely essential for the inhibitory activity. These results were supported by experiments using a recombinant form of the corresponding pig von Ebner's gland protein (VEGp). This protein, which does not possess a fully conserved first cystatin-like motif, is unable to inhibit papain.     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Hydrophobic binding domains of rat intestinal maltase-glucoamylase

Rat intestinal microvillus maltase-glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Sephadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Sepharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with ethanol--formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26-28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase-glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

A hydrophobic form of the small-intestinal sucrase-isomaltase complex

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in the tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.     

A hydrophobic form of the small-intestinal sucrase-isomaltase complex.

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.     

Evidence for the existence of a multichain proteoglycan of heparan sulphate

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.     

Isolation of antiserum against rat transcortin and characteristics of specific antibodies

Repeated chromatography of rat plasma protein on DEAE-cellulose, hydroxylapatite and subsequent gel-filtration through Sephadex G-200 were used to obtain a pure rat transcortin homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of transcortin was about 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Immunization of a rabbit with the homogeneous preparation of rat plasma transcortin caused development of antibodies to transcortin. It was shown that the antibodies of rabbit antisera in the experiments made in vitro and in vivo neutralized 60 and 65% of 3H-corticosterone-transcortin complexes, respectively. Specific antibodies to the transcortin were isolated from the homogeneous fraction of IgG by affinity chromatography on transcortin-sepharose 4B. 125J-labelled antibodies were adsorbed by protein A-sepharose; IgG can be eluted by IM acetic acid as a sharp peak. The SDS-polyacrylamide gel electrophoresis demonstrated that affinity-eluted material contains 25 and 50 kDa polypeptides.     

A new protein with a particular thermoprecipitability in bovine milk.

A new protein with a particular thermoprecipitability was isolated from bovine milk and tentatively termed milk pyroglobulin. The protein which was soluble at a relatively cold temperature was precipitated by raising the temperature to a certain degree depending on the concentration of the protein. The precipitate disappeared on recooling. This protein had the electrophoretic mobility of gamma globulin but did not carry either antigenic specificities of immunoglobulins or of free secretory component. The molecular weight was estimated to be approximately 60,000 in thin-layer gel filtration on Sephadex G-200 superfine gel, but the protein appeared to be convertible to molecules with a lower molecular weight of approximately 20,000 in the presence of bovine serum albumin. The presence of the albumin inhibited the thermoprecipitation as did alpha-lactalbumin but not IgG immunoglobulin from bovine colostrum. In SDS-polyacrylamide gel electrophoresis, the protein was separated into two components having a molecular weight of 19,000 and 10,000, respectively. Both components were thermoprecipitable and carried identical antigenic determinants.     

Tumor-associated blocking factors: isolation from sera of tumor-bearing mice.

An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.     

Preparation and properties of sialomucopolysaccharides obtained from rat brain

Sialomucopolysaccharides were released from the defatted protein residue by the proteolytic action of papain after extraction of rat whole brains with chloroform-methanol (2:1, v/v). Further purification is achieved by dialysis to remove low-molecular-weight fragments and by precipitation of nucleic acids and glucuronic acid-containing mucopolysaccharides by treatment with cetylpyridinium chloride. Gel filtration of the sialomucopolysaccharides through Sephadex G-200 removes the major portion of the impurities that absorb light in the ultraviolet region. The sialomucopolysaccharides were fractionated on DEAE-Sephadex to yield a population of sialomucopolysaccharides that show an increase in N-acetylneuraminic acid content and a decrease in fucose content as the concentration of chloride required to elute the individual components is increased. On gel filtration on Sephadex G-200, those sialomucopolysaccharide molecules rich in N-acetylneuraminic acid and poor in fucose appear to be larger molecules than those rich in fucose and poor in N-acetylneuraminic acid. A structure is proposed in which all sialomucopolysaccharide molecules are assumed to possess the same repeating unit consisting of hexosamine and hexose. The molecules differ from each other in the number of fucose and N-acetylneuraminic acid residues attached to the basic structure. Most of the hexosamine is present as glucosamine, although one fraction was obtained that appeared to contain galactosamine. Most of the hexose present is accounted for as galactose and mannose, although small amounts of glucose were found in some fractions. Methods of analysis for the N-acetylneuraminic acid and hexosamine components of the sialomucopolysaccharides were defined.     

Sizes of isotopically labeled molecules released during lysis of tumor cells labeled with 51Cr and [14C]nicotinamide.

Previous studies of mechanisms of immune lysis have utilized the release of isotopically labeled molecules from cells labeled with Na₂⁵¹Cr0₄ and [¹⁴C]nicotinamide, among others. The interpretation of specific isotope release from such cells was hampered by the lack of knowledge about the molecular sizes of the released radioactive molecules. In the present study, P815 tumor cells were labeled with the above two isotopic compounds. The cells were then lysed by freezing and thawing, antibody and complement, or immune T-lymphocyte-mediated killing. Soluble supernatants from the lysates were chromatographed in the cold on Sephadex G-10, G-25, and G-200 using several elution media. No differences were detected due to different kinds of lysis or different eluting media. Eighty-five percent of ¹⁴C-labeled molecules released from the cells were indistinguishable from nicotinamide and clearly distinct from nicotinamide adenine dinucleotide. The results suggest that [¹⁴C]nicotinamide taken up by the tumor cells during overnight culture is released unaltered during lysis. Ninety percent of ⁵¹Cr-labeled lysate molecules had an apparent molecular weight less than 4000. Most of this material was distinct from inorganic chromate, but its chemical form is not known.     

Papain solubilization of the Epstein-Barr virus-induced membrane antigen.

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) was successfully solubilized from the membranes of viable EBV-infected Raji cells by treatment with papain (5 to 6 U per 1 X 10(7) to 2 X 10(7) cells). The loss of MA from viable cells was monitored by membrane immunofluorescence and antibody-dependent cellular cytotoxicity. Soluble MA was demonstrated in papain digests through inhibition of antibody-dependent cellular cytotoxicity and by inhibition of the binding of anti-MA antibodies to cells as detected by use of 125I-labeled staphylococcal protein A. Approximately 75% of the MA activity in the extracts was not sedimentable at 100,000 X g,, indicating that the majority of EBV MA activity that was released by this procedure was associated with small-molecular-weight material. Antiserum prepared from an owl monkey immunized with these papain extracts contained antibody to MA and neutralizing antibodies, but lacked detectable antibodies against viral capsid antigens and EBV-induced early antigens.     

Induction of gamma-glutamyl transpeptidase by hexachlorocyclohexane

Hexachlorocyclohexane (BHC) induced gamma-Glutamyl transpeptidase in rat liver. The enzyme was partially purified from normal BHC fed and fetal liver and also from hepatoma. The gel filtration and electrophoretic properties of the BHC-induced enzyme was compared against that of the other three. Chemical induced hepatoma showed an additional peak of activity in Sephadex G-200 filtration. The other enzymes could be cleaved by papain to give a fraction which cochromatographed with the additional peak of hepatoma enzyme. BHC-induced enzyme and normal enzyme had similar electrophoretic mobility but differed from that of hepatoma and fetal liver enzyme which showed a slightly slower movement.     

Inhibition of human collagenase activity by a small molecular weight serum protein.

Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α₂-macroglobulin and a smaller serum component which eluted after α₁-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α₁-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α₁-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.