scholarly journals Analysis of the protein and glycan parts of CA125 antigen from human amniotic fluid

2007 ◽  
Vol 59 (2) ◽  
pp. 97-103 ◽  
Author(s):  
Bojana Milutinovic ◽  
Miroslava Jankovic
Keyword(s):  
Amniotic Fluid ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Placental Tissue ◽  
Peptide Mass Fingerprinting ◽  
Chain Structure ◽  
Exchange Chromatography ◽  
Molecular Forms ◽  
Human Amniotic Fluid ◽  
Peptide Mass

CA125 antigen is a mucin-type molecule with a complex protein backbone and oligosaccharide chain structure. In this study, we characterized CA125 antigen from human amniotic fluid by gel filtration, ion-exchange chromatography, peptide mass fingerprinting and lectin-binding assays. The obtained results indicate CA125 to be structurally heterogeneous, existing in different glycoisoforms with subtle differences in the profile of molecular forms in comparison to placental tissue-derived and cancer-derived CA125 antigen. The complexity of CA125 structure suggests that it can act as a multifunctional molecule. Further investigation is therefore needed in order for the biological meaning of the tissue-specific structural forms to be comprehended fully. .

scholarly journals Molecular forms and activities of glycosidases in cultures of amniotic-fluid cells

1976 ◽  
Vol 155 (3) ◽  
pp. 599-605 ◽  
Author(s):  
B Hultberg ◽  
J Lindsten ◽  
S Sjöblad
Keyword(s):  
Ion Exchange ◽  
Amniotic Fluid ◽  
Lysosomal Enzymes ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molecular Forms ◽  
Cell Type ◽  
Amniotic Fluid Cells ◽  
Total Enzyme

Ion-exchange chromatography of gel filtration demonstrated the presence of different molecular forms of nine lysosomal enzymes in cultured amniotic-fluid cells. The patterns of molecular forms were similar to those known from skin fibroblasts and liver tissue. During cultivation total enzyme activities fluctuated with the number of passages, without any consistent trend of increase or decrease, and without correlation to the dominating cell type in the culture.

scholarly journals Prostaglandin D2 synthase: a component of human amniotic fluid and its association with fetal abnormalities

1996 ◽  
Vol 42 (7) ◽  
pp. 1042-1050 ◽  
Author(s):  
D N Melegos ◽  
H Yu ◽  
E P Diamandis
Keyword(s):  
Amniotic Fluid ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Prostaglandin D2 ◽  
Amino Acid Sequence Analysis ◽  
Human Amniotic Fluid ◽  
Immunoreactive Band ◽  
The Brain ◽  
Renal Abnormalities

Abstract Prostaglandin D2 (PGD2) synthase is responsible for PGD2 production in the brain. Western blot analysis of human amniotic fluid and probing with a polyclonal antibody against prostate-specific antigen (PSA) revealed a strong immunoreactive band with a molecular mass of 25 kDa. The immunoreactive species, which does not react with monoclonal anti-PSA antibodies, was purified to homogeneity from 1 L of amniotic fluid through successive cycles of gel filtration and ion-exchange chromatography. Amino acid sequence analysis (15 cycles) revealed that the protein was highly homologous or identical to PGD2 synthase. On semiquantitative analysis, PGD2 synthase concentration appears to increase dramatically during gestational weeks 12-25 and then declines slowly until term. PGD2 synthase concentration in amniotic fluid was altered in many abnormal pregnancies, most notably its decrease in trisomic fetuses and fetuses with renal abnormalities.

The Nature of Inactive Renin in Human Plasma and Amniotic Fluid

1978 ◽  
Vol 55 (1) ◽  
pp. 41-50 ◽  
Author(s):  
A. A. Shulkes ◽  
R. R. Gibson ◽  
S. L. Skinner
Keyword(s):  
Molecular Weight ◽  
Amniotic Fluid ◽  
Human Plasma ◽  
Gel Filtration ◽  
Dilution Effect ◽  
Exchange Chromatography ◽  
Acid Activation ◽  
Temperature Dependent ◽  
Inactive Renin ◽  
Full Activation

1. The properties of inactive and active renin in human plasma and amniotic fluid were studied chromatographically. Activation was achieved at pH 3.3 with and without added pepsin. 2. Acid activation of renin was time- and temperature-dependent but was inhibited by dilution of the sample. The dilution effect was corrected by adding pepsin. Such characteristics indicate that activation at low pH is catalysed by intrinsic enzymes. 3. Separation and/or dilution of the activating enzyme during ion-exchange chromatography concealed the eluted position of inactive renin and reduced the amount recovered. Only after full activation of the eluted renin was achieved with added pepsin was a distinct peak of inactive renin exposed. 4. At pH 7.5 inactive renin carried a lower negative charge than the active enzyme. This charge difference was lost after activation. 5. No molecular-weight differences between active, inactive renin or the International Renin Standard were detected by gel filtration. No renin of larger molecular weight was present. 6. These findings will be helpful in purification studies of human inactive renin.

scholarly journals Collagenous constituents of amniotic fluid.

1998 ◽  
Vol 45 (4) ◽  
pp. 1037-1046
Author(s):  
T Gogiel ◽  
D A Bielecki ◽  
E Bańkowski
Keyword(s):  
Amniotic Fluid ◽  
Classical Method ◽  
Degradation Products ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Monomeric Form ◽  
Alpha Subunits ◽  
Bacterial Collagenase ◽  
Sds Page ◽  
Collagen Degradation Products

The amniotic fluid (AF) was fractionated by dialysis, gel filtration and SDS/PAGE, and submitted to the assay of collagenous constituents. The collagenous character of peptides and proteins of amniotic fluid was confirmed by hydroxyproline (Hyp) assay and treatment with bacterial collagenase followed by electrophoresis and gel filtration of the digestion products. It was found that AF contains collagen degradation products but the classical method of Hyp determination described by Woessner (Arch. Biochem. Biophys., 1961, 93, 440-447) gives overestimated values due to the interference with other AF components. Fractionation of AF on Sephadex G-100 column allowed to remove the interfering material and to estimate the actual Hyp content which equals to approx. 6.2 microg/ml. About 70% of Hyp was found in low molecular dialyzable products and the rest (about 30%) appears to be a constituent of nondialyzable collagenous polypeptides of the molecular mass of about 7.9-26.3 kDa. It is suggested that such collagenous polypeptides may be the products of proteolytic conversion of collagen precursor (procollagen) into the monomeric form of this protein. No high molecular forms of collagen, corresponding to alpha-subunits, were found.

scholarly journals 2,3-Dihydroxypropane-1-sulfonate degraded by Cupriavidus pinatubonensis JMP134: purification of dihydroxypropanesulfonate 3-dehydrogenase

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1556-1564 ◽  
Author(s):  
Jutta Mayer ◽  
Thomas Huhn ◽  
Michael Habeck ◽  
Karin Denger ◽  
Klaus Hollemeyer ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Peptide Mass Fingerprinting ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Major Facilitator Superfamily ◽  
Bacterial Degradation ◽  
Uptake System ◽  
Peptide Mass ◽  
Terrestrial Environments ◽  
Sulfoacetaldehyde Acetyltransferase

2,3-Dihydroxypropane-1-sulfonate (DHPS) is a widespread intermediate in plant and algal transformations of sulfoquinovose (SQ) from the plant sulfolipid sulfoquinovosyl diacylglycerol. Further, DHPS is recovered quantitatively during bacterial degradation of SQ by Klebsiella sp. strain ABR11. DHPS is also a putative precursor of sulfolactate in e.g. Ruegeria pomeroyi DSS-3. A bioinformatic approach indicated that some 28 organisms with sequenced genomes might degrade DHPS inducibly via sulfolactate, with three different desulfonative enzymes involved in its degradation in different organisms. The hypothesis for Cupriavidus pinatubonensis JMP134 (formerly Ralstonia eutropha) involved a seven-gene cluster (Reut_C6093–C6087) comprising a LacI-type transcriptional regulator, HpsR, a major facilitator superfamily uptake system, HpsU, three NAD(P)+-coupled DHPS dehydrogenases, HpsNOP, and (R)-sulfolactate sulfo-lyase (SuyAB) [EC 4.4.1.24]. HpsOP effected a DHPS-racemase activity, and HpsN oxidized (R)-DHPS to (R)-sulfolactate. The hypothesis for Roseovarius nubinhibens ISM was similar, but involved a tripartite ATP-independent transport system for DHPS, HpsKLM, and two different desulfonative enzymes, (S)-cysteate sulfo-lyase [EC 4.4.1.25] and sulfoacetaldehyde acetyltransferase (Xsc) [EC 2.3.3.15]. Representative organisms were found to grow with DHPS and release sulfate. C. pinatubonensis JMP134 was found to express at least one NAD(P)+-coupled DHPS dehydrogenase inducibly, and three different peaks of activity were separated by anion-exchange chromatography. Protein bands (SDS-PAGE) were subjected to peptide-mass fingerprinting, which identified the corresponding genes (hpsNOP). Purified HpsN converted DHPS to sulfolactate. Reverse-transcription PCR confirmed that hpsNOUP were transcribed inducibly in strain JMP134, and that hpsKLM and hpsNOP were transcribed in strain ISM. DHPS degradation is widespread and diverse, implying that DHPS is common in marine and terrestrial environments.

scholarly journals Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by Syntrophomonas wolfei

2009 ◽  
Vol 191 (19) ◽  
pp. 6167-6177 ◽  
Author(s):  
Nicolai Müller ◽  
David Schleheck ◽  
Bernhard Schink
Keyword(s):  
Electron Transport ◽  
Coenzyme A ◽  
Peptide Mass Fingerprinting ◽  
Exchange Chromatography ◽  
Triphenyltetrazolium Chloride ◽  
Oxidoreductase Activity ◽  
Cell Extracts ◽  
Peptide Mass ◽  
Syntrophomonas Wolfei ◽  
Reversed Electron Transport

ABSTRACT Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H2 generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.

scholarly journals Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds ofBorreria hispidaon Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
S. Rupachandra ◽  
D. V. L. Sarada
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Protein Fraction ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Peptide Mass Fingerprinting ◽  
Dependent Manner ◽  
Peptide Mass ◽  
Hela Cell Lines ◽  
Apoptotic Activity

A 35 KDa protein referred to as F3 was purified from the seeds ofBorreria hispidaby precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase ofPyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds ofBorreria hispidawith antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells.

scholarly journals Cel9D, an Atypical 1,4-β-d-Glucan Glucohydrolase from Fibrobacter succinogenes: Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases

2008 ◽  
Vol 190 (6) ◽  
pp. 1976-1984 ◽  
Author(s):  
Meng Qi ◽  
Hyun-Sik Jun ◽  
Cecil W. Forsberg
Keyword(s):  
Biomass Conversion ◽  
Peptide Mass Fingerprinting ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Endoglucanase Activity ◽  
Catalytic Residues ◽  
Peptide Mass

ABSTRACT The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to β-1→4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k cat /K m values for cello-oligosaccharides with higher degrees of polymerization. The k cat /K m value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-β-d-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.

scholarly journals Molecular forms of human prostate-specific antigen in urine of subjects with benign prostatic hyperplasia

2006 ◽  
Vol 58 (2) ◽  
pp. 77-82 ◽  
Author(s):  
Maja Kosanovic ◽  
Miroslava Jankovic
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Structural Complexity ◽  
Exchange Chromatography ◽  
Prostatic Hyperplasia ◽  
Molecular Forms ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity

In the present study, we examined mollecular forms of urinary prostate-specific antigen (PSA), focusing on its structural complexity in general and specifically on its microheterogeneity in relation to benign prostatic hyperplasia (BPH). Gel filtration, ion-exchange chromatography, and lectin-affinity chromatography were used to characterize PSA. In comparing the binding pattern of PSA isoforms, moderate changes were observed in the relative abundance of distinct molecular subpopulations separated on lectin-affinity columns. They may be related to alteration in the position and type of linkage of fucose or sialic acid, as well as to modification of the trimannosyl core by branching of the PSA oligosaccharide chain.

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