scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

scholarly journals Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

1997 ◽  
Vol 326 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Sergio LIZANO ◽  
Bruno LOMONTE ◽  
Jay W. FOX ◽  
José Maréa GUTIÉRREZ
Keyword(s):  
Phospholipase A2 ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Biological Activities ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Cytolytic Activity ◽  
Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

scholarly journals Purification and characterization of thyroid transcription factor 2

1994 ◽  
Vol 304 (3) ◽  
pp. 981-985 ◽  
Author(s):  
D Civitareale ◽  
A Saiardi ◽  
P Falasca
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Thyroid Tissue ◽  
Dna Binding Protein ◽  
Purification And Characterization ◽  
Thyroid Transcription Factor

Thyroid transcription factor 2 binds to the promoters of both thyroglobulin and thyroperoxidase genes, two markers of thyroid tissue differentiation, and its binding modulates the activity of both promoters. In this paper we describe the purification of thyroid transcription factor 2 essentially to homogeneity and demonstrate that it is a thyroid-specific DNA-binding protein. Furthermore, we provide a biochemical characterization suggesting that thyroid transcription factor 2 binds to DNA as a dimer and that it is a zinc-finger DNA-binding protein regulated in vitro by the redox state.

Effects of cigarette smoke condensate on the production and characterization of exopolysaccharides by Streptococcus mutans / Sigara dumanı kondensatının Streptococcus mutans tarafından üretilen eksopolisakkaritlerin üretimi ve karakterizasyonu üzerine etkisi

2016 ◽  
Vol 41 (4) ◽  
Author(s):  
Chun ping Xu ◽  
Ying Zeng ◽  
Hongqian Shentu ◽  
Aijing Zhao ◽  
Duobin Mao
Keyword(s):  
Molecular Mass ◽  
Molecular Characterization ◽  
Streptococcus Mutans ◽  
Cigarette Smoke ◽  
Molecular Conformation ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Cigarette Smoke Condensate ◽  
Acetic Acid Production

AbstractObjective: Streptococcus mutans is regarded as the major agent causing dental caries. It has been well documented that cigarette smoke affects the growth of S. mutans. This study investigate the effect of cigarette smoke condensate (CSC) on the production and characterization of exopolysaccharides (EPS) produced by S. mutans ATCC 35668.Methods: Cigarettes of Shanhua brand were used to prepare the CSC. S. mutans was cultured in MSB media with the addition of CSC under anaerobic condition. Furthermore, the EPS fraction was isolated and purified by gel filtration chro-matography on Sepharose CL-6B. The molecular characterization of EPS was analyzed by GC-MS, FT-IR and size exclusion chromatography/multiangle laser light scattering (SEC/MALLS) system.Results: The results showed that CSC at tested concentrations could significantly increase the growth of S. mutans and acetic acid production, compared with the control. The CSC was not found to affect carbohydrate composition of the EPS, but the molecular mass of EPS decreased from 3.04×104 g/mol (without CSC) to 2.75×104 (with CSC). The SEC/MALLS also revealed the molecular conformation of EPS changed from flexible coil to globular shape in aqueous solution.Conclusion: This study revealed that CSC was directly able to affect molecular mass and structural conformation of EPS from S. mutans. The molecular characterization of EPS would become an indicator in certain pathological disorders.

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

2001 ◽  
Vol 356 (1) ◽  
pp. 257-267 ◽  
Author(s):  
Jeffrey A. SKINNER ◽  
Alan R. SALTIEL
Keyword(s):  
Molecular Mass ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Terminal Region ◽  
Protein Phosphatase 1 ◽  
Chromosome 15 ◽  
Glutathione S Transferase ◽  
Interacting Protein

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

scholarly journals Detection of Macroprolactinemia and Molecular Characterization of Prolactin Isoforms in Blood Samples of Hyperprolactinemic Women

2012 ◽  
Vol 31 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Rajinder Chawla ◽  
Tibebe Antonios ◽  
Esaiyas Berhanu ◽  
Gonfa Ayana
Keyword(s):  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Cross Sectional Study ◽  
Sectional Study ◽  
Cross Sectional ◽  
Blood Samples ◽  
High Prevalence ◽  
Prolactin Isoforms

Detection of Macroprolactinemia and Molecular Characterization of Prolactin Isoforms in Blood Samples of Hyperprolactinemic WomenProlactin (PRL) circulates in the blood in the form of monomeric prolactin, dimeric prolactin and macroprolactin. Macroprolactin is a common cause of hyperprolactinemia. The objective of this study was to assess the prevalence of macroprolactinemia in hyperprolactinemic women and to undertake the biochemical characterization of macroprolactin. A retrospective cross-sectional study was conducted on one hundred hyperprolactinemic patients. All the sera were subjected to polyethylene glycol (PEG) precipitation and were divided into true hyperprolactinemics (PRL recovery >60%), probable macroprolactinemics (PRL recovery between 40 and 60%) and macroprolactinemics (PRL recovery < 40%). The prevalence of macroprolactinemia was found to be 34%. Sera from each group were further analyzed for isoforms of prolactin by gel filtration chromatography (GFC). The clinical spectrum of presenting complaints in the hyperprolactinemic cohort included oligomenorrhea, galactorrhea and infertility, but the presentation did not differ between macroprolactinemic and truly hyperprolactinemic patients. GFC showed three major PRL isoforms, viz., 23.5 kDa (monomeric), 47 kDa (dimeric) and 150-174.6 kDa (PRL-IgG complexes) along with the medium and heavy weight aggregates of prolactin. The results of the study showed that macroprolactinemia is one of the causes of hyperprolactinemia with high prevalence. It is recommended that all hyperprolactinemic patients be screened for macroprolactinemia.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Identification of Potential Key lncRNAs in the Context of Mouse Myeloid Differentiation by Systematic Transcriptomics Analysis

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 630
Author(s):  
Yongqing Lan ◽  
Meng Li ◽  
Shuangli Mi
Keyword(s):  
Transcription Factor ◽  
Cell Model ◽  
Myeloid Differentiation ◽  
Rna Seq ◽  
Hematopoietic Differentiation ◽  
Granulocytic Differentiation ◽  
Expression Of Genes ◽  
Non Coding Rnas

Hematopoietic differentiation is a well-orchestrated process by many regulators such as transcription factor and long non-coding RNAs (lncRNAs). However, due to the large number of lncRNAs and the difficulty in determining their roles, the study of lncRNAs is a considerable challenge in hematopoietic differentiation. Here, through gene co-expression network analysis over RNA-seq data generated from representative types of mouse myeloid cells, we obtained a catalog of potential key lncRNAs in the context of mouse myeloid differentiation. Then, employing a widely used in vitro cell model, we screened a novel lncRNA, named Gdal1 (Granulocytic differentiation associated lncRNA 1), from this list and demonstrated that Gdal1 was required for granulocytic differentiation. Furthermore, knockdown of Cebpe, a principal transcription factor of granulocytic differentiation regulation, led to down-regulation of Gdal1, but not vice versa. In addition, expression of genes involved in myeloid differentiation and its regulation, such as Cebpa, were influenced in Gdal1 knockdown cells with differentiation blockage. We thus systematically identified myeloid differentiation associated lncRNAs and substantiated the identification by investigation of one of these lncRNAs on cellular phenotype and gene regulation levels. This study promotes our understanding of the regulation of myeloid differentiation and the characterization of roles of lncRNAs in hematopoietic system.

scholarly journals Molecular characterization of Saccharomyces cerevisiae Δ3,Δ2-enoyl-CoA isomerase. Vol. 273 (1998) 33184–33191

2005 ◽  
Vol 280 (13) ◽  
pp. 13203
Author(s):  
Brian V. Geisbrecht ◽  
Dai Zhu ◽  
Kerstin Schulz ◽  
Katja Nau ◽  
James C. Morrell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Characterization
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