scholarly journals Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast

1994 ◽  
Vol 304 (2) ◽  
pp. 407-415 ◽  
Author(s):  
M Wilson ◽  
M Sullivan ◽  
N Brown ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Molecular Mass ◽  
Protein A ◽  
Affinity Column ◽  
Camp Phosphodiesterase ◽  
Human Type ◽  
Native Molecular Mass ◽  
Type Iv ◽  
Apparent Homogeneity ◽  
Specificity Constant

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.

scholarly journals Purification and characterization of a connective-tissue-degrading metalloproteinase from the cytosol of metastatic melanoma cells

1987 ◽  
Vol 245 (2) ◽  
pp. 429-437 ◽  
Author(s):  
S Zucker ◽  
T Turpeenniemi-Hujanen ◽  
N Ramamurthy ◽  
J Wieman ◽  
R Lysik ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Type I Collagen ◽  
Collagen Type I ◽  
Enzymic Activity ◽  
Affinity Column ◽  
Type I ◽  
Type Iv ◽  
Affinity Column Chromatography ◽  
Molecular Masses

A metalloproteinase with activity against type IV collagen, type I collagen and gelatin has been purified from the cytosol of a highly metastatic mouse melanoma by anion-exchange, zinc-chelated and lectin-affinity column chromatography. The purified enzyme has a molecular mass of approx. 59 kDa and on isoelectric focusing in two-dimensional gels produced three spots with apparent isoelectric points (pI) between 5.7 and 6.1. Enzymic activity with collagen, but not gelatin, substrates was latent, requiring activation by trypsin or organomercurials. Trypsin activation of this metalloproteinase was accompanied by a change in molecular mass, whereas autoactivation after 1 month's storage, was not. The degradation of types I and IV collagen by the melanoma enzyme yielded products of lower molecular masses than those yielded by mammalian collagenases, this characteristic thus differentiating this metalloproteinase from classical collagenases.

scholarly journals Characterization of sheep lacrimal-gland peroxidase and its major physiological electron donor

1996 ◽  
Vol 314 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Abhijit MAZUMDAR ◽  
Ratna CHATTERJEE ◽  
Subrata ADAK ◽  
Anil GHOSH ◽  
Chhabinath MONDAL ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Electron Donor ◽  
Lacrimal Gland ◽  
Coupled System ◽  
Antigenic Determinants ◽  
Compound I ◽  
Native Molecular Mass ◽  
Apparent Homogeneity ◽  
Sigmoidal Curve

A soluble sheep lacrimal-gland peroxidase was purified to apparent homogeneity. It had a native molecular mass of 75 kDa with a subunit molecular mass of 82 kDa and an isoelectric point of 6.5. Western blotting showed that it shares some of the enzyme antigenic determinants in common with other soluble peroxidases. The enzyme exhibits a Soret peak at 410 nm which is shifted to 431 nm by 5 equiv. of H2O2 due to the formation of compound II. The latter is, however, unstable and gradually returns to the native state. The enzyme forms complexes with CN- and N3- and is reduced by dithionite showing a characteristic reduced peroxidase spectrum. Although the enzyme oxidizes I-, SCN- and Br- optimally at pH 5.5, 5.25 and 5.0 respectively, at physiological pH, it oxidizes I- and SCN- only. Since extracellular SCN- concentration is much higher than I-, SCN- may act as the major electron donor to the enzyme. The second-order rate constants for the reaction of the enzyme with H2O2 (k+1) and of compound I with SCN- (k+2) were 4×107 M-1·s-1 and 8.1×105 M-1·s-1 respectively. A plot of log Vmax. against pH yields a sigmoidal curve consistent with a single ionizable group on the enzyme with a pKa value of 5.75, controlling thiocyanate oxidation. In a coupled system with the peroxidase, H2O2, SCN-, GSH, NADPH and glutathione reductase, peroxidase-catalysed SCN- oxidation by H2O2 could be coupled to NADPH consumption. The system is proposed to operate in vivo for the efficient elimination of endogenous H2O2.

Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

1990 ◽  
Vol 48 (3) ◽  
pp. 944-945
Author(s):  
Ichiro Yamamoto ◽  
Toshiaki Tachibana ◽  
Hiroko Maruyama ◽  
Noriyuki Komatsu ◽  
Hiroyuki Kuramoto ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Cell Lines ◽  
Endometrial Adenocarcinoma ◽  
Protein A ◽  
Rabbit Antiserum ◽  
Carcinoma Cells ◽  
Affinity Column ◽  
Antibody Technique ◽  
Galactosyl Transferase ◽  
C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

scholarly journals Virulence Factors Found in Nasal Colonization and Infection of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates and Their Ability to Form a Biofilm

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 14
Author(s):  
Thamiris Santana Machado ◽  
Felipe Ramos Pinheiro ◽  
Lialyz Soares Pereira Andre ◽  
Renata Freire Alves Pereira ◽  
Reginaldo Fernandes Correa ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Sccmec Type ◽  
Protein A ◽  
Invasive Infection ◽  
Type I ◽  
Methicillin Resistant ◽  
Type Iv ◽  
Spa Gene ◽  
The City

Hospitalizations related to Methicillin-resistant Staphylococcus aureus (MRSA) are frequent, increasing mortality and health costs. In this way, this study aimed to compare the genotypic and phenotypic characteristics of MRSA isolates that colonize and infect patients seen at two hospitals in the city of Niterói—Rio de Janeiro, Brazil. A total of 147 samples collected between March 2013 and December 2015 were phenotyped and genotyped to identify the protein A (SPA) gene, the mec staphylococcal chromosomal cassette (SCCmec), mecA, Panton-Valentine Leucocidin (PVL), icaC, icaR, ACME, and hla virulence genes. The strength of biofilm formation has also been exploited. The prevalence of SCCmec type IV (77.1%) was observed in the colonization group; however, in the invasive infection group, SCCmec type II was prevalent (62.9%). The Multilocus Sequence Typing (MLST), ST5/ST30, and ST5/ST239 analyses were the most frequent clones in colonization, and invasive infection isolates, respectively. Among the isolates selected to assess the ability to form a biofilm, 51.06% were classified as strong biofilm builders. Surprisingly, we observed that isolates other than the Brazilian Epidemic Clone (BEC) have appeared in Brazilian hospitals. The virulence profile has changed among these isolates since the ACME type I and II genes were also identified in this collection.

scholarly journals Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

1990 ◽  
Vol 265 (10) ◽  
pp. 5840-5846
Author(s):  
M M Van Lookeren Campagne ◽  
E Wu ◽  
R D Fleischmann ◽  
M M Gottesman ◽  
K W Chason ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Mammalian Cells ◽  
Camp Phosphodiesterase

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

Functionally distinct isoforms of the CRE-BP DNA-binding protein mediate activity of a T-cell-specific enhancer

1992 ◽  
Vol 12 (2) ◽  
pp. 747-757
Author(s):  
K Georgopoulos ◽  
B A Morgan ◽  
D D Moore
Keyword(s):  
T Cell ◽  
Dna Binding ◽  
Cyclic Amp ◽  
Protein A ◽  
Cell Receptor ◽  
Protein Isoforms ◽  
Cdna Clones ◽  
C Terminus ◽  
Transcription Regulators ◽  
Cyclic Amp Response Element

Expression of the CD3 delta gene of the T-cell receptor (TCR) complex is regulated by a T-cell-specific enhancer. A highly conserved 40-bp motif (element delta A) within the CD3 delta enhancer is responsible for mediating its activity and specificity. Element delta A exhibits sequence similarities to the cyclic AMP response element (CRE) but does not respond to changes in the level of cyclic AMP. Using the delta A element as a probe, we have isolated three cDNA clones encoding three distinct protein isoforms, products of differential splicing and alternate promoter usage of the CRE-BP gene. These isoforms share the DNA binding and dimerization domains at the C terminus of the protein but differ at their N termini. In transfection assays, their activities as transcription regulators differ: CRE-BP2 is a potent activator, CRE-BP3 is a weak activator, and CRE-BP1 is transcriptionally inert. Mutations in the basic region of the CRE-BP1 protein which abrogate its ability to bind DNA render this protein a dominant repressor of the delta A enhancer. Antibodies to the CRE-BP protein interact specifically with the ubiquitous and predominantly T-cell-restricted nuclear complexes that bind to the delta A element and suggest the presence of this protein in homo- and heterodimeric complexes. Since the delta A motif is also present in the enhancer and promoter of the TCR alpha and beta genes, the CRE-BP isoforms may mediate expression of other members of the CD3/TCR complex during T-cell development.

scholarly journals Mass spectrometric analysis of rat liver cytosolic glutathione S-transferases: modifications are limited to N-terminal processing

1995 ◽  
Vol 308 (1) ◽  
pp. 69-75 ◽  
Author(s):  
H I Yeh ◽  
C H Hsieh ◽  
L Y Wang ◽  
S P Tsai ◽  
H Y Hsu ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Female Rats ◽  
Spectrometric Analysis ◽  
Affinity Column ◽  
Female Rat ◽  
Significant Difference ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses ◽  
Rat Livers

Cytosolic glutathione S-transferases (GSTs) from rat livers were purified using an S-hexylglutathione affinity column. The GST subunits were resolved by reverse-phase HPLC and their molecular masses were determined by electrospray mass spectrometry. The major hepatic GSTs detected were subunits 1, 1′, 2, 3 and 4, with molecular mass of 25,520, 25,473, 25,188, 25,782 and 25,571 Da respectively. Subunits 6, 7 and 10 are minor components, with molecular mass of 25,551, 23,308 and 25,211 Da respectively. Alternatively, the hepatic GSTs were purified using a glutathione affinity column. Subunits 1, 1′, 2, 8 and 10 were eluted from this column with GSSG, the oxidized form of glutathione. Subunit 8 has a molecular mass of 25,553 Da. The remaining proteins on the glutathione affinity column were removed with glutathione and S-hexylglutathione. Subunits 2, 3, 4 and 6 could be detected in the eluate. We could not detect any significant difference in molecular mass between GSTs isolated from male and female rat livers. Cytosolic GSTs were isolated from livers of buthionine sulphoximine-treated female rats for MS analysis. The molecular masses obtained were identical to those determined for the controls.

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