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Query: UMLS:C0272170 (SDS)
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A method for the rapid manual isolation of polytene chromosomes and nuclear membranes from salivary glands of Chironomus tentans is presented and the analysis of some of their RNA and protein components before and after treatment with 2 M salt solutions is summarized.--After salt-incubation the chromosomes still display a considerable number of bands which stain with ethidium bromide and which are sensitive to treatment with DNase, RNase, trypsin, and proteinase K, to a lesser extent with pronase and papain. Analysis of the iodinated residual proteins on SDS gels yield three major and two minor bands (MW between 50,000 and 70,000 dalton) which were also shown to be present in interphase chromosomes of Ehrlich ascites cells which had been treated similarly and are also tightly bound constituents of DNA prepared according to Gross-Bellard et al. (1973). This result indicates the existence of a general class of non-histone proteins involved in keeping the DNA in a supercoiled state. Furthermore their presence in salt-treated nuclear membranes of Chironomus salivary gland cells (and Xenopus oocytes, unpubl.) will be of interest with respect to functional aspects of the nuclear matrix.
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PMID:Effect of salt-treatment on manually isolated polytene chromosomes from Chironomus tentans. 35 13

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.
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PMID:Characterization of two species of Fc fragments obtained from rat IgG2a by prolonged papain digestion. 40 15

Recent studies of the micron- and kappa-chains of the first patient (GLI) with micronHCD indicated that the observed defect was the result of the failure of assembly of the intact kappa-chain to the micron-chain, which lacked the VH domain but had the CH1 Cys normally linked to the light chain. To explore the possibility that the VH region is necessary for the formation of the HL disulfide bond, in vitro studies were performed with GLI micron- and kappa-chains and with the CH1 domain and kappa-chain derived from an IgG3 myeloma protein, KUP, which yields separate VH, CH1, and kappa-chains after papain digestion and reduction. The proteins were reduced and allowed to reoxidize, and the combination products were assessed by gel chromatography under dissociating conditions by SDS-PAGE and by immunoprecipitation techniques. The results suggest that, although in vitro covalent and noncovalent combinations are possible between intact light chains and their autologous heavy chains even in the absence of the VH domain, the efficiency is less than that when the intact Fd region is used. Hence, it seems likely that lack of VH alone is not sufficient to explain the failure of assembly observed in muHCD.
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PMID:The interaction of immunoglobulin heavy and light chains in the absence of the VH domain. 40 2

Mannose-binding hemagglutinins were found in the extracts of a pyocyanin-forming Pseudomonas aeruginosa, which contain galactose-specific hemagglutinins. They were purified simultaneously with the latter proteins by heating to 70 degrees C, precipitating with ammonium sulfate, application to a Sepharose 4B column, and elution from it by 0.05 M mannose. The mannose-specific hemagglutinins were shown to be similar to the galactophilic ones in (a) being glycoproteins of very low molecular weight (about 11 000 by SDS gel electrophoresis), (b) their tendency to aggregate, and (c) their ability to effect stronger agglutination of erythrocytes treated with papain than of untreated ones. They were found to resemble them also in their reaction with simple sugars and interactions with divalent cations, which are essential for their activity. In these properties, as well as in their relative resistance to heat and to proteolytic enzymes, these two types of bacterial hemagglutinins are like most of the plant, contrasted with the animal, hemagglutinins. The reactions with mannose and mannose-bearing compounds (yeast mannan, horseradish peroxidase (EC 1.11.1.7), and serum globulins), which are not shared with the galactophilic Pseudomonas hemagglutinins, indicate a relationship of the mannose-binding protein of Pseudomonas to the plant lectin concanavalin A. The mannose-binding hemagglutinins do not exhibit identical cell-agglutinating spectra owing to difference in profiles of sugar specificity and relative affinity to mannose derivatives compared with free mannose.
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PMID:Mannose-binding hemagglutinins in extracts of Pseudomonas aeruginosa. 40 63

Antibodies, raised against pure renin from the submaxillary gland of mice, were used to obtain renin specific Fab fragments. The purification steps were DEAE-chromatography, followed by papain digestion with separation of the undigested IgG preparation from the Fab/Fc fragments on a Sephadex G-100 column. Finally the Fab fragments were subjected to affinity chromatography on a CH-Sepharose 4B column with submaxillary renin attached. The purified Fab fragments revealed only a single band in SDS-polyacrylamide gel electrophoresis and a single precipitation line in cross immunoelectrophoresis. The association constants for the reaction of renin with the purified Fab fragments compared to the divalent antibodies were of the same magnitude, 0.7 x 10(11) l/mol and 1.0 x 10(11) l/mol, respectively. Comparison of the affinity of the Fab fragments for the antigenic determinants and the enzymatic inhibition of renin were determined to be approximately the same. Thus, the pure specific immunoreactive Fab fragment of antirenin, with an inhibitor constant of 1.5 x 10(-11), is the most potent inhibitor of mouse renin, so far.
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PMID:Purification and characterization of rabbit anti-mouse renin specific Fab fragments. 44 51

The protein moiety of squid (Watasenia scintillans) rhodopsin has been shown to have a molecular weight of 46 800 by means of amino acid analysis. This value was comparable to the value (51 000) obtained from SDS-polyacrylamide gel electrophoresis. After the squid eyes were incubated at 10 degrees C for 8 days, the rhodopsin showed a molecular weight of 39 000 on electrophoresis. The smaller molecular weight was ascertained by amino acid analysis of the rhodopsin; and may result from autolysis by the lysosomal enzyme. The rhodopsin in rhabdomeric membranes and in detergent solution was treated with chymotrypsin, papain or subtilisin. These enzymes first produced the 39 000 dalton rhodopsin and then cleaved this into the 25 000 and 14 000 dalton peptides without bleaching. The rhodopsin was attacked by proteases and readily lost an approx. 12 000 dalton peptide portion. This portion included the COOH-terminal and was rich in glutamic acid, proline, glycine, alanine and tyrosine residues.
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PMID:Molecular weight and structural studies on cephalopod rhodopsin. 46 26

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.
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PMID:Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate. 48 55

Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.
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PMID:Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle. 53 1

The aragonite crystals in the molluscan bivalve hinge ligament are surrounded by an organic sheath which is distinct from the remainder of the ligament matrix. Methods have been developed to isolate these sheathed crystals from the ligaments of Spisula solidissima and Mercenaria mercenaria employing a papain digestion of the matrix protein. The sheathed crystals from Spisula have a CaCO3/protein ratio of 11.1 and those from Mercenaria a ratio of 29.6. The sheathed crystals and the empty crystal sheaths have been examined by electron microscopy for structural integrity. The sheath proteins exhibit much smaller proportions of the amino acids glycine and methionine than the hinge ligaments. These are characteristic amino acids of high concentrations in the hinge ligaments of both species. The concentrations of acidic and basic amino acids are increased about two fold in the sheaths over those of the ligaments. Otherwise there is little similarity in the amino acid composition of the sheaths in the two species. However, SDS electrophoresis shows the sheaths of both to contain a major protein component with a molecular weight of about 25,000. The sheath protein from the Mercenaria ligament contains about 5% carbohydrate and that of Spisula sheaths less than 1% carbohydrate.
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PMID:The crystal sheaths from bivalve hinge ligaments. 64 36

Procedures were developed for isolating highly purified cytoplasmic granules of basophilic leukocytes from guinea pig peripheral blood. The methods involved disruption of cells in 0.34 M sucrose followed by a series of membrane filtrations and fractionation on sucrose density gradients. These preparations, up to 95% pure basophil granules by electron microscopy, contained a mixture of neutral esterases-proteases including caseinolytic activity; both trypsin- and chymotrypsin-like serine hydrolases were identified by means of appropriate inhibitors. Localization of at least one such activity to the basophil granule was confirmed by a cytochemical method; this activity was absent in contaminating lymphocytes and eosinophils. By contrast, several lysosomal enzymes, lactic dehydrogenase, and plasminogen activator activity, present in cell homogenates, were absent from purified granules. The granule matrix of guinea pig basophils, unlike the cytoplasmic granules of other granulocytes or mast cells, was little altered by high or low salt concentration but was disrupted into insoluble fragments by 0.01 N HCl and by Triton X-100. Granules were solubilized by papain and by urea-SDS but enzyme activity was destroyed. Triton X-100 incubation with freeze-thawing proved to be the optimal method for extracting esterase activities. Esterase activities were not released from basophils under conditions of anaphylactic degranulation that liberated the great majority of basophil granule histamine.
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PMID:Isolation of the cytoplasmic granules of guinea pig basophilic leukocytes: identification of esterase and protease activities. 87 25


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