scholarly journals THE SEPARATION AND CHARACTERIZATION OF SUBMICROSCOPIC COMPONENTS OF HEPATIC CYTOPLASM

1959 ◽  
Vol 7 (2) ◽  
pp. 126-132 ◽  
Author(s):  
MITURU TAKANAMI
Keyword(s):  
Alkaline Phosphatase ◽  
Principal Components ◽  
Biochemical Properties ◽  
Sedimentation Rates ◽  
Differential Centrifugation ◽  
Ultrasonic Oscillation ◽  
Ribonucleoprotein Complexes ◽  
Acid And Alkaline Phosphatase

In an ultracentrifugal study on the cytoplasmic supernatant of rabbit liver, the following two principal components were separated from the supernatant by differential centrifugation and their biochemical properties investigated: (1) a granular substance sedimented at a rate of about 250s (250s component) and (2) a few macromolecular components the sedimentation rates of which were roughly in the range of from 40s to 100s (macromolecular components). The 250s component, which was rich in lipids and easily disintegrated into smaller units by treatments of ultrasonic oscillation and of Nadesoxycholate, exhibited much higher activities of dipeptidase, acid and alkaline phosphatase than the macromolecular components. By contrast, the latter macromolecular components which belonged to ribonucleoprotein complexes exhibited comparatively high activities of RNase and esterase. Uptake in vivo of radioactive phosphate (P32) by the RNA contained in the above two principal components markedly differed from each other. When the RNA contained in the 250s component was separated by the use of Nadesoxycholate into RNA in a non-sedimentable portion and that in a sedimentable portion corresponding to a ribonucleoprotein coplex, the RNA in the latter state showed an uptake rate extremely different from that of the macromolecular components. So it is emphasized that the ribonucleoprotein complex comprised in the 250s component and that existing free in the cytoplasm (i.e. macromolecular components) are metabolically different.

Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

scholarly journals Characterization of the Yersinia enterocolitica Type III Secretion ATPase YscN and Its Regulator, YscL

2006 ◽  
Vol 188 (10) ◽  
pp. 3525-3534 ◽  
Author(s):  
Bill Blaylock ◽  
Kelly E. Riordan ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Atp Hydrolysis ◽  
Biochemical Properties ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Type Iii ◽  
Bacterial Proteins

ABSTRACT Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

1973 ◽  
Vol 19 (10) ◽  
pp. 1225-1233 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Sodium Dodecyl ◽  
Current Theory ◽  
Outer Cell ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
A Subunit

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.

Chlamydomonas reinhardtiiproduces a profilin with unusual biochemical properties

2001 ◽  
Vol 114 (23) ◽  
pp. 4293-4305 ◽  
Author(s):  
David R. Kovar ◽  
Pinfen Yang ◽  
Winfield S. Sale ◽  
Bjørn K. Drobak ◽  
Christopher J. Staiger
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cell Body ◽  
Mating Type ◽  
Immunofluorescence Microscopy ◽  
Adenine Nucleotide ◽  
Biochemical Properties ◽  
Primary Role ◽  
Displacement Assay

We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably the only profilin isoform, is present in both the cell body and flagella. Examination of vegetative and gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment of CrPRF at the anterior of the cell near the base of flagella and near the base of the fertilization tubule in mating type plus gametes. Purified, recombinant CrPRF binds to actin with a Kd value ∼10–7 and displaces nuclei in a live cell ‘nuclear displacement’ assay, consistent with profilin’s ability to bind G-actin in vivo. However, when compared with other profilin isoforms, CrPRF has a relatively low affinity for poly-L-proline and for phosphatidylinositol (4,5) bisphosphate micelles. Furthermore, and surprisingly, CrPRF inhibits exchange of adenine nucleotide on G-actin in a manner similar to human ADF or DNase I. Thus, we postulate that a primary role for CrPRF is to sequester actin in Chlamydomonas. The unusual biochemical properties of CrPRF offer a new opportunity to distinguish specific functions for profilin isoforms.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

scholarly journals Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

2014 ◽  
Vol 197 (5) ◽  
pp. 1002-1011 ◽  
Author(s):  
Annika Flint ◽  
Alain Stintzi
Keyword(s):  
Escherichia Coli ◽  
Campylobacter Jejuni ◽  
Biochemical Properties ◽  
Wild Type ◽  
Visible Spectroscopy ◽  
Content Type ◽  
Increased Sensitivity ◽  
Uv Visible

Catalase enzymes detoxify H2O2by the dismutation of H2O2into O2and H2O through the use of hemin cofactors. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content inCampylobacter jejunicatalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol194:334–345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interactin vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed inEscherichia coli. The importance of tyrosine 57 in hemin traffickingin vivowas confirmed by introducing thecj1386Y57Aallele into aC. jejuniΔcj1386mutant background. Thecj1386Y57Amutation resulted in increased sensitivity toward H2O2relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the Δcj1386+cj1386Y57Amutant had significantly reduced hemin content compared to that of thecj1386WTbackground. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

1989 ◽  
Vol 63 (4) ◽  
pp. 315-327 ◽  
Author(s):  
Takahiro Fujino ◽  
Koichi Fukuda ◽  
Fusanori Hamajima ◽  
Yoichi Ishii
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Phosphatase Activity ◽  
Metabolic Activity ◽  
Acid And Alkaline Phosphatase ◽  
Excretory Bladder ◽  
Histochemical Tests ◽  
Parenchymal Cells

ABSTRACTHistochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.

scholarly journals Microbiological and biochemical properties of an agricultural Mexican soil amended with sewage sludge

2012 ◽  
Vol 36 (5) ◽  
pp. 1646-1655 ◽  
Author(s):  
Renato Armenta ◽  
Rocio Vaca ◽  
Jorge Lugo ◽  
Pedro del Aguila
Keyword(s):  
Alkaline Phosphatase ◽  
Organic Matter ◽  
Sewage Sludge ◽  
Microbial Biomass ◽  
Soil Microbial Biomass ◽  
Biochemical Properties ◽  
Basal Respiration ◽  
Soil Microbial ◽  
Acid And Alkaline Phosphatase

The application of sewage sludge is a concern because it may affect the quality of organic matter and microbiological and biochemical soil properties. The effects of surface application of sewage sludge to an agricultural soil (at 18 and 36 t ha-1 dry basis) were assessed in one maize (Zea mays L.) growing season. The study evaluated microbial biomass, basal respiration and selected enzymatic activities (catalase, urease, acid and alkaline phosphatase, and β-glucosidase) 230 days after sewage sludge application and infrared spectroscopy was used to assess the quality of dissolved organic matter and humic acids. Sewage sludge applications increased the band intensity assigned to polysaccharides, carboxylic acids, amides and lignin groups in the soil. The organic matter from the sewage sludge had a significant influence on the soil microbial biomass; nevertheless, at the end of the experiment the equilibrium of the soil microbial biomass (defined as microbial metabolic quotient, qCO2) was recovered. Soil urease, acid and alkaline phosphatase activity were strongly influenced by sewage sludge applications.

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