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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sodium dodecylsulphate precipitates very low density lipoproteins (VLDL and chylomicrons) rich in triglyceride and a narrow correlation (r = 0.9) was found between the turbidimetric index SDS and triglyceridemia. Heparin in calcium medium acts in the same way and precipitates also low density lipoproteins (LDL) rich in cholesterol. A correlation (r = 0.875) was drawn up between the cholesterol LDL and the difference between the turbidimetric indices (heparin Ca -- SDS). In the first case, we were thus measuring by turbidimetry a VLDL + chylomicron index and, in the second case, an LDL index which permits one to obtain simplified typing of the hyperlipoproteinemias. A nomogram linking the two indices of hyperlipoproteinemia type was drawn up experimentally. This orientation analysis may be completed by electrophoresis on polyacrylamide gel used in concentration gradient and pH. In the system proposed, the lipoproteins were previously stained with tetrazolium nitroblue and clearly shown up. The chylomicrons in particular, become separated from the VLDL, the sinking pre-beta-lipoprotein or Lp (a) was identifiable and the type III hyperlipemia was easily diagnosed.
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PMID:[Analysis of lipoproteins using polyacrylamide gel electrophoresis and fractionated precipitation with polyanions and detergents. Use in the classification of hyperlipoproteinemias]. 18 41

The delta-subspecies of protein kinase C (PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose Fast Flow, Phenyl 5PW, Heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was doublet with molecular weight of 78 kDa and 76 kDa on SDS-PAGE. This doublet proteins were separated partially by Mono Q column chromatography, both of which were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78 kDa protein was a phosphorylated form of the 76 kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid, and was purified for comparison. This recombinant enzyme was also doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mapping. In addition, these the enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, and Ca2+. Comparison was also made between the enzymological properties of delta PKC and alpha PKC, such as activation kinetics, sensitivity to protein kinase inhibitors and substrate specificity which were distinctly different from each other.
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PMID:Enzymatic properties of ubiquitously expressed delta-subspecies of protein kinase C differing from other members of protein kinase C family. 129 10

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.
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PMID:Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence. 133 88

Utilizing three proteins plus tyrosine-glutamate copolymer as substrates, all of which are subjected to (near) stoichiometrical phosphorylation exclusively on tyrosine residues, we partially purified four different protein-tyrosine phosphatases (PTPases) from rat liver cytosol which differed in substrate preference. Of the four PTPases, tentatively termed L1, L2, L3, and L4, PTPase L1 was purified to apparent homogeneity by a procedure involving chromatography on DEAE-cellulose at pH 7.0, Blue Sepharose, DEAE-cellulose at pH 7.6, hydroxyapatite, Phenyl Sepharose, Mono Q, and TSKgel Heparin. PTPase L1 was purified about 7000-fold from the extract and 0.27 mg was isolated from 1000 g liver corresponding to a yield of 13% from the Blue Sepharose step where it had become freed from any other PTPases detectable by our assay procedure. The purified PTPase L1 showed a major protein band of 67 kDa on SDS/PAGE. Catalytically, PTPase L1 had a specific activity of about 6500 nmol Pi released min-1mg-1 toward tyrosine-glutamate copolymer phosphorylated on tyrosine residues. PTPase L1 exhibited very low sensitivities to PTPase inhibitors such as zinc acetate, sodium vanadate, and acidic compounds as compared with those of most of the PTPases purified thus far. Amino acid sequence analysis of the purified PTPase L1 revealed a partial peptide sequence showing similarity to the catalytic domain core sequences conserved in the PTPase family. PTPase L1 was most similar to a PTPase termed PTP1C encoded by a human breast carcinoma cDNA but the identity was 55% over 117 residues spanning nearly half of the catalytic domain of PTP1C. The analysis also revealed another partial peptide sequence (113 residues) 70% identical with the sequence corresponding to 68% of two adjacent copies of the src homology region 2(SH-2 domain) identified in PTP1C. Besides those peptide sequences, PTPase L1 had regional sequences which were 70-90% identical with the residues lying between the two SH-2 domains or between the more C-terminal SH-2 domain and the catalytic domain of the carcinoma PTPase.
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PMID:Purification and characterization of a rat liver protein-tyrosine phosphatase with sequence similarity to src-homology region 2. 138 83

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.
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PMID:A DNA polymerase from maize axes: its purification and possible role. 146 49

The substances extracted and partially purified from embryos of the loach (Misgurnus fossilis L.) at the late blastula stage are shown to possess properties characteristic of the fibroblast growth factor (FGF): 1) they bind with Heparin-Sepharose; 2) induce DNA synthesis in mouse NIH-3T3 and Swiss-3T3 fibroblast cell lines and in the primary culture of human embryonic fibroblasts; 3) have an apparent molecular weight of 15-16 kDa (according to SDS-electrophoresis); and 4) show positive reaction with antibodies against bovine basic FGF.
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PMID:[The detection and characteristics of substances with fibroblast growth factor properties in the cells of loach embryos]. 149 56

Tyrosine kinase activity was studied in the crude cytosolic and particulate fraction of normal mucosa and squamous cell carcinomas of the upper aero-digestive tract. In the presence of exogenously added phosphorylation substrate (Glu,Tyr4:1), the cytosolic tyrosine kinase activity was 6-fold higher in tumors compared to normal mucosa (p = 0.001), and in the particulate fraction the increase was 8-fold in tumors compared to normal mucosa. Different proposed tyrosine kinase inhibitors, including genistein, quercetin and the alpha-cyanocinnamide ST 638, were tested for their ability to inhibit phosphorylation of the synthetic tyrosine phosphorylation substrate. Phosphorylation of Glu,Tyr4:1 in tumors (cytosolic fraction) was reduced to 77.8 +/- 8.7% of the control value by 10 microM ST 638 (p less than 0.05), and to 50.7 +/- 10.4% by 100 microM quercetin (p less than 0.01). In normal mucosa (cytosolic fraction) the corresponding values were 41.7 +/- 16.6% in the presence of 10 microM ST 638 (p less than 0.05) and 32.1 +/- 5.8% in the presence of 100 microM quercetin (p less than 0.05). These inhibitors had no effect on the tyrosine kinase activity in the particulate fractions. Phosphorylation of endogenous proteins in the crude cytosolic fraction was evaluated by SDS-polyacrylamide gel electrophoresis after alkali treatment of the gels. Autoradiography of the gels treated in this manner revealed bands corresponding to phosphorylated proteins with apparent molecular weight of 18, 23, 37-38, 42-44 (double band), 53-55 (double band), 61 and 92-94 (double band) kD. Quercetin (100 microM) markedly reduced the phosphorylation of these proteins, while no effect of ST 638 could be seen. Heparin (20 micrograms/ml) stimulated the phosphorylation of three proteins with apparent molecular weight of 39 and about 72 kD, respectively, and inhibited the phosphorylation of 2 proteins with molecular weight of 92 and 53 kD in tumors. These features were observed in both tumors and normal tissue, with the exception that heparin only stimulated the 72 kD band in normal mucosa and that the phosphorylation was markedly higher in tumors. In summary, our results show an increased tyrosine phosphorylation in squamous cell carcinomas of the upper aero-digestive tract compared to normal oral mucosa. These differences and their origin might be of vital importance in the regulation of events leading to malignant transformation.
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PMID:Tyrosine kinase activities in normal and neoplastic epithelia tissue of the human upper aero-digestive tract. 181 86

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or HC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.
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PMID:Mechanism of potentiation of antithrombin III and heparin cofactor II inhibition by sulfated xylans. 197 2

An intracellular form of phospholipase A2 was purified about 47,500-fold to near homogeneity from bovine platelets 100,000 x g supernatant by sequential use of column chromatographies on Heparin-Sepharose, DEAE-Sephacel, Butyl-Toyopearl, Sephacryl S-300, DEAE-5PW HPLC, TSK G 3000 SW HPLC and Mono Q FPLC. The final preparation showed a single band on SDS-polyacrylamide gel, and its molecular mass was estimated to be approximately 100,000 daltons. The purified PLA2 showed maximal activity at alkaline pH(pH 9.0-10.0) and considerable activity at 0.3-1.0 microM calcium concentration. It hydrolyzed phosphatidylcholine containing arachidonate at sn-2 position with high selectivity in comparison to linoleate.
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PMID:Purification and some properties of a phospholipase A2 from bovine platelets. 198 99

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.
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PMID:Purification and characterization of heparin-binding growth factors from porcine uterus. 231 Mar 77


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