Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial endotoxin (lipopolysaccharide, LPS) induces coagulation of horseshoe crab hemolymph. Our previous studies had demonstrated that a hemolymph factor, designated factor B, was associated with the LPS-mediated activation of the Limulus clotting system [Ohki et al. (1980) FEBS Lett. 120, 318-321]. On further purification of factor B we found that an additional component, designated factor C, was required to generate factor B activity in the presence of LPS in order to activate the proclotting enzyme. To elucidate the role of factor C in the LPS-mediated reaction, factor C was isolated and characterized from the hemocyte lysate under sterile conditions. The preparation exhibited a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while two protein bands on SDS-PAGE were observed after reduction. Thus, factor C had a Mr of 123 000 consisting of a heavy chain of Mr = 80 000 and a light chain of Mr = 43 000. Factor C was converted to an activated form in the presence of LPS with a Mr = 123 000, designated factor C. Upon activation, cleavage of the light chain occurred resulting in the accumulation of two new fragments of Mr = 34000 and 8500 on reduced SDS-PAGE. A diisopropylfluorophosphate-sensitive active site was localized in the light chain (Mr = 34000) of factor C. The reconstitution experiments, using factor C, factor B, proclotting enzyme and LPS, demonstrated that all of these proteins are essential for the endotoxin-mediated coagulation system. On the basis of these results we propose that a cascade pathway of LPS-induced activation of the Limulus clotting system consists of three sequential activations of hemolymph serine protease zymogens.
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PMID:Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Isolation and characterization. 351 66

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.
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PMID:Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes. 351 94

Chicken and turkey beta 2-m were isolated from citrated plasma in sequential use of three chromatographic steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta 2-m by reaction with a beta 2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta 2-m and 4.7 and 4.8 for turkey beta 2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta 2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. EM280 was 15.9 for chicken beta 2-m and 16.4 for turkey beta 2-m. The amino acid compositions and sequences of the two avian beta 2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N-terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta 2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.
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PMID:Isolation and characterization of chicken and turkey beta 2-microglobulin. 354 90

Cathepsin B was purified from rabbit, human, ox and sheep liver. SDS-polyacrylamide gel electrophoresis after reaction of the purified cathepsin B samples with the active site directed inhibitor, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido- ([3H]acetamido)-butane ([3H]Ac-Ep-459), showed that the enzyme exists as either a two-chain form (approx. Mr 25,000 and 4,000), a single-chain form (approx. Mr 30,000) or both. The active site was found in the light chain of the two-chain forms. The two-chain and single-chain forms of ox cathepsin B were separated by ion exchange chromatography and shown to have similar catalytic activities against the substrates Z-Phe-Arg-4-methyl-7-coumarylamide (Z-Phe-Arg-NHMec) and Z-Arg-Arg-NHMec and rates of inhibition by L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4- guanidino)butane (E-64). Cathepsin L was purified from the same four species, and compared with the enzyme from rat liver. SDS-polyacrylamide gel electrophoresis after reaction of the purified cathepsin L preparations with the active site directed inhibitor, [3H]Ac-Ep-459, showed that cathepsin L from each species consists of two chains; a light chain of approx. Mr 5,000 and a heavy chain of approx. Mr 25,000, which contained the active site cysteine. All species variants of cathepsin L were recognized by the antibody to the human enzyme. With Z-Phe-Arg-NHMec as substrate, kinetic constants were found to be similar for all five species (Km 1-4 microM and and kcat 10-30 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Species variations amongst lysosomal cysteine proteinases. 355 68

When DNP-SH1-myosin, selectively dinitrophenylated at SH1 by 1,2,4-trinitrobenzene, was irradiated with a high-pressure mercury lamp equipped with a UV cut filter, a new 220-kDa band called the X-band appeared right above the heavy chain band (200 kDa) on SDS-PAGE (Laemmli). The time course of the X-band formation was composed of two phases, the initial one being rapid, and the second slow. Immune reaction experiments using antibodies specific for heavy or light chains indicated that the X-band in the initial phase contained heavy chain alone, but no light chains. Such an extra band (106 kDa) was also observed in the initial phase of photolysis of DNP-SH1-Subfragment-1 (heavy chain: 96 kDa) obtained from DNP-SH1-myosin. Trypsinolysis of the 106-kDa product generated a 83-kDa band. N-Terminal sequence analysis and the amino acid composition of the band revealed that the X-band is an intraheavy chain cross-linking product between the 20- and the 50-kDa fragments. This presents a striking contrast to the other cross-linking from SH1 using benzophenone-4-iodoacetamide which reacted with the 25-kDa fragment alone (Lu, R.C. et al. (1986) Proc. Natl. Acad. Sci. U.S. 83, 6392-6396). Based upon the result obtained, the spatial arrangement of the three tryptic domains around SH1 is discussed.
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PMID:Photocross-linking from DNPated SH1 in myosin head. I. Cross-linking to the 50-kDa fragment. 359 45

The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.
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PMID:The regulation of membrane-bound and secreted immunoglobulins in the human lymphoid cell line LICR-LON and human hybridomas. 365 99

Lectin selectivity for human Ig classes is based on carbohydrate differences. Earlier reports that the lectin jacalin precipitated human IgA were confirmed and supplemented by the current study, which demonstrates that jacalin also binds human IgD as evaluated by micro-ELISA and SDS-PAGE. Experimental findings indicated that: (i) Monoclonal and polyclonal (sera) IgD, IgA1, but not IgA2, IgM, or IgG1-4 reacted with jacalin. (ii) Six tested monoclonal IgD proteins each bound approximately equally to jacalin when antigenicity rather than protein concentration was measured: the results weigh against the presence of jacalin-detectable IgD subclasses or genetic variants. (iii) IgD and IgA1 both associated maximally in 4-8 h at 4 degrees C. There was no dissociation at 4 degrees C but limited dissociation occurred at 37 degrees C after 24 h. (iv) Both IgD and IgA1 were eluted from jacalin by galactose-related sugars. (v) IgD and IgA1 bind competitively to jacalin. The results suggested that jacalin reacts with O-linked oligosaccharide N-acetyl-galactosamine (GalN) residues found on the hinge region of both IgD and IgA1. Jacalin also interacted with one major and several minor unidentified sera proteins. The findings offer an approach to the isolation of serum polyclonal IgD and to the characterization of the unusual carbohydrates of the human delta heavy chain with respect to their function.
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PMID:Human IgD and IgA1 compete for D-galactose-related binding sites on the lectin jacalin. 365 39

Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes. Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.
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PMID:Isolation and partial characterization of adenylate cyclase-enriched membranes from human platelets. 371 86

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti-light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.
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PMID:Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies. 373 Jun 10

Rat immunoglobulin E (IgE) synthesized in Xenopus laevis oocytes, injected with rat plasmacytoma mRNA, was analysed by specific immunoprecipitation and SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The results indicate that the oocytes will translate and correctly process the rat IgE heavy and light chains, resulting in secretion of a correctly assembled, normal immunoglobulin molecule. The normal, extensive glycosylation of the IgE heavy chain (e-chain) is faithfully carried out by the oocytes; therefore, this posttranslational modification is apparently of an unspecific nature, and does not depend upon a mechanism specific for plasma cells.
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PMID:Synthesis, processing, and secretion of rat immunoglobulin E made in Xenopus oocytes. 378 Sep 73


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