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)

In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, thrombin, and glycosaminoglycans in more detail. Inhibition of thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 microgram/mL, significantly promoted thrombin inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal inhibition in the presence of heparin. Virtually no inhibition of thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 microgram/mL.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Specific glycosaminoglycans support the inhibition of thrombin by plasminogen activator inhibitor 1. 843 48

Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells in vitro and an angiogenic factor in vivo. Its role in other cell types is not yet clear. To explore its possible involvement in malignant transformation, we studied the expression of its receptors in normal and malignant melanocytes. Binding and cross-linking experiments showed that human melanoma cells but not normal melanocytes express VEGF receptors. Separation of reaction products by SDS-PAGE demonstrated the presence of 125I-VEGF/receptor complexes of 180 and 165 kDa in the melanoma cells. A diffuse complex with a mass of approximately 235 kDa was also detected in some experiments. Heparin enhanced the binding of the radioactive ligand to the receptors of the WW94 and SW1614 melanoma cell lines. This binding was completely abolished by heparinase digestion and was restored by the addition of exogenous heparin, indicating that heparin-like molecules are necessary for ligand/receptor interaction. This study suggests that the aberrant expression of VEGF receptors is one of the phenotypic changes occurring in melanoma cells during malignant transformation.
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PMID:Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors. 843 21

The signals that trigger the cytodifferentiation of oligodendrocytes (OLGs) are largely unknown. Using as a model system cultures of pure OLGs, we have shown that adhesion to a substratum initiates myelinogenesis (Yim SH, Szuchet S, Polak PE, J Biol Chem 261:11808-11815, 1986). It was of interest to investigate whether components such as proteoglycans (PGs) play any role in the biology of OLGs as it pertains to myelinogenesis. We set out to determine first, whether OLGs carry PGs; second, the nature of the association of these components with OLG plasma membrane; and third, if and how these PGs are modulated by OLG-substratum interaction. We compared the expression and characteristics of PGs extracted with different solvents from nonattached (B3.f) and attached (B3.fA) OLGs. B3.f and B3.fA OLG cultures were labeled with carrier-free 35SO4(2-) in serum-free medium. After removing excess label, OLGs were treated with heparin to extract susceptible components. Pellets were then exposed to 1% Triton X-100 plus 0.1 M NaCl and subsequently to 4 M guanidine-HCl plus 0.5 M NaCl. Solutions containing extracted material were characterized by size-exclusion chromatography, SDS-PAGE, and enzymatic degradation. Herein we report that (1) OLGs display [35S]PGs on their surface within 24 hr of substratum adhesion, and (2) these PGs can be operationally classified as peripheral and integral. We further show that the peripheral PGs are of high and intermediate size as assessed by size-exclusion chromatography and are segregated within the plasma membrane in such a way that the species with intermediate mass are extracted while OLGs remain adhered, whereas the high-molecular-weight species are only extracted after OLGs have been detached. Heparin also dislodges a number of sulfated proteins/Gps. Only a single class--high molecular weight--of integral PGs was identified; this PG requires guanidine-HCl for extraction. All PGs belong to the heparan sulfate class as evidenced by their degradation with heparitinase and their lack of susceptibility to chondroitinase ABC. The common theme of our findings is that these macromolecules have basal levels of expression in the nonadhered OLGs but undergo an adhesion-induced enhancement in their syntheses. We postulate that these PGs (1) play a role in OLG-substratum adhesion and hence myelinogenesis, and (2) may be determinants in establishing OLG polarity. Such polarization is the first overt sign of OLG functional differentiation and occurs prior to any morphological differentiation, e.g., extension of processes does not occur until 48 hr later when the plasma membrane is already polarized.
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PMID:Oligodendrocyte proteoglycans: modulation by cell-substratum adhesion. 847 42

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.
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PMID:Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III. 848 44

The data are presented about isolation from the brain of 7.5-day chick embryos of a factor capable of neuralizing effect on the early gastrula ectoderm of the grass frog Rana temporaria L. Earlier this factor was defined as embryonic brain-derived neuralizing factor (EBDNF) (Mikhailov, Gorgoliuk, 1989). The isolation procedure includes (1) extraction with deionized water at pH 9.0; (2) ion exchange chromatography on a column with DEAE-adsorbent at pII 8.0; (3) affinity chromatography on Heparin-Ultragel column. EBDNF-containing fraction is eluted from the Heparin-Ultragel column with 250 mM NaCl as a separate peak. Four bands are observed on SDS-electrophoregrams of this fraction, two more prominent ones having the molecular weight of 43 and 63 kDa. The yield of EBDNF-containing fraction is about 0.01-0.02% of the wet weight of the initial brain tissue.
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PMID:[The embryonic brain-derived neuralizing factor (EBDNF): its partial purification by ion-exchange and affinity chromatography]. 848 10

We tried to purify pregnancy-associated plasma protein A (PAPP-A) from normal term maternal serum in this study by means of a three step chromatographic procedure. At first 30 ml of maternal serum samples were applied to a column for sieve chromatography and eluted according to molecular weight. PAPP-A was detected in the high molecular weight fraction area by radioimmunoassay for PAPP-A, and 91% of the total amount of PAPP-A in the maternal samples was recovered. Secondary PAPP-A containing fractions were applied to a Heparin-Sepharose column and eluted by a stepwise increase in NaCl 0.15, 0.30 and 0.60 M in 0.05 M Tris-HCl buffer, pH 7.8. Ninety-three percent of PAPP-A in applied samples was recovered under the 0.6 M NaCl condition. Thirdly the pooled fractions which contained PAPP-A after Heparin-Sepharose affinity chromatography were applied to DEAE-Sephacel Chromatography and eluted by a stepwise increase in NaCl 0.15, 0.30 and 0.45 M in 0.01 M acetate buffer, pH 5.5. Ninety-one percent of PAPP-A in applied samples was recovered under the 0.3 M NaCl condition. Finally PAPP-A containing fractions were concentrated 10 times and 3.8 IU (1.7 mg) of PAPP-A was isolated. The purification schedule removed approximately 99% of total protein in the maternal serum while 74% of PAPP-A was recovered. The purification factor (fold), which was calculated as the increase in specific activity (mIU:PAPP-A/mg:protein) in comparison with the starting value in the maternal serum, was 526. And the purity (mg:PAPP-A/mg:protein) of the final product was 68.4%. Analysis of the final purified material by SDS-PAGE showed a single band of 200kDa, and western blot analysis showed that the main purified protein was PAPP-A. The immunological identity of the PAPP-A purified in this study to the PAPP-A donated by Teisner et al., was recognized by crossed immunoelectrophoresis and tandem crossed immunoelectrophoresis. We hope that the new PAPP-A purified in this study can be utilized for the development of a sensitive and convenient assay for PAPP-A and its standard material, and also for basic study to clarify the biological function of PAPP-A in the human body.
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PMID:[Isolation and purification of pregnancy-associated plasma protein A]. 857 17

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.
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PMID:Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing. 860 4

A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.
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PMID:Purification and characterization of a 58,000-Da proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi. 875 95

Human chorionic gonadotropin (hCG) in first trimester placental cells is composed of immature alpha- and beta-subunits containing only N-linked high-mannose sugar chains. Intracellular immature intermediates are accumulated in rough endoplasmic reticulum in much greater quantity than mature hCG composed of mature subunits. We have previously shown that this immature hCG might be bound to other protein(s), including an ATP-binding protein, forming high molecular weight-hCG (HMW-hCG), which is not aggregate of immature hCG alone. To identify the ATP-binding protein forming the HMW-hCG in detail, proteins in HMW-hCG preparation were photoaffinity-labeled with 8-azido-[alpha-32P]ATP. Autoradiography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the labeled protein with M(r) = 78000 was immunoprecipitated with any antibody against alpha-subunit, beta-subunit and hCG, indicating that this protein is bound to immature hCG. Furthermore, to determine whether some other proteins associate to form HMW-hCG, we purified HMW-hCG without breakdown to its components using columns of DE52, Heparin-Sepharose and Sephacryl S-300. As the final step of the purification, HMW-hCG was allowed to adsorb on a column of ATP-agarose and anti-hCG IgG-agarose, respectively. SDS-PAGE analysis of eluted proteins from the columns bound to the respective column via the constituent of HMW-hCG, such as ATP-binding protein or immature hCG, showed four common protein bands with molecular weights of 78000, 43000, 28000 and 20000. The protein with M(r) = 43000 was stained with any antibody against alpha-subunit, beta-subunit and hCG, indicating it to be immature hCG. The protein band with M(r) = 78000, which might correspond to the ATP-binding protein described above, was stained with anti-heat shock protein 70 (HSP70) monoclonal antibody. To confirm the association of immature hCG and HSP70-like protein, immature hCG preparation was incubated with HSP70-like protein purified from placental extracts. The molecular weight change of immature hCG appeared to increase by this incubation and was close to HMW-hCG, but not exactly the same. These results suggest that immature hCG intermediate exists as HMW-hCG containing HSP70-like protein, which has ATP-binding capacity, and two other proteins in first trimester placental cells.
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PMID:Purification and characterization of high molecular weight hCG from human first trimester placenta. 878 79

Selenoprotein Ph, the human analogue of selenoprotein P from rat plasma, was purified from human plasma using Heparin Sepharose chromatography, PEG precipitation, DEAE ion exchange chromatography. RP chromatography, SDS-PAGE and electroelution. SDS-PAGE of the purified protein revealed one broad-selenium containing protein band from 56 to 67 kDa with a selenium maximum at 62 kDa. Using a 7.5% T gel this band was separated into two distinct selenium-containing bands with molecular weights of 61 and 64 kDa.
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PMID:Improved procedure for the purification of selenoprotein Ph from human plasma. 884 59


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