scholarly journals Tyrosine phosphate hydrolysis of host proteins byTrypanosoma cruziis linked to cell invasion

1998 ◽  
Vol 161 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Li Zhong ◽  
Hong-Gang Lu ◽  
Silvia N.J Moreno ◽  
Roberto Docampo
Keyword(s):  
Cell Invasion ◽  
Host Proteins ◽  
Phosphate Hydrolysis ◽  
Tyrosine Phosphate ◽  
Hydrolysis Of

scholarly journals Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jennifer Timm ◽  
Maria Valente ◽  
Daniel García-Caballero ◽  
Keith S. Wilson ◽  
Dolores González-Pacanowska
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Trypanosoma Brucei ◽  
Cell Invasion ◽  
Essential Amino Acids ◽  
Host Cell Invasion ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

Alkaline phosphatases of Neurospora crassa. Part II product inhibition studies

1972 ◽  
Vol 18 (4) ◽  
pp. 407-421 ◽  
Author(s):  
F. W. J. Davis ◽  
Howard Lees
Keyword(s):  
Neurospora Crassa ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Phosphate Hydrolysis ◽  
Non Linear ◽  
Inhibition Studies ◽  
Enzyme Complexes ◽  
Hydrolysis Of

A partially purified preparation of the constitutive alkaline phosphatase from Neurospora crassa, containing two electrophoretically distinct activities was used in initial studies of product inhibition patterns. Inorganic phosphate was shown to be a linear competitive inhibitor, and p-nitrophenol to be a non-linear, non-competitive inhibitor of p-nitrophenyl phosphate hydrolysis. Glycerol was shown to be a linear non-competitive inhibitor of β-glycerophosphate hydrolysis.A purification procedure whereby one enzyme activity could be obtained free of the second was devised. The purified enzyme catalyzed the hydrolysis of a wide range of substrates and had a molecular weight of 111 000. Its hydrolysis of glucose 6-phosphate was competitively inhibited by phosphate and non-competitively inhibited by glucose. Both inhibitions were linear. Hydrolysis of p-nitrophenyl phosphate was competitively inhibited by phosphate in a linear manner, but p-nitrophenol was a non-linear, non-competitive inhibitor. Alternate product inhibition by glucose was linear competitive. No inhibition by p-nitrophenol of glucose 6-phosphate hydrolysis could be detected.The inhibition data for glucose 6-phosphate and β-glycerophosphate may be consistent with an ordered Uni-Bi mechanism expanded to include one or more isomerizations of enzyme complexes. The postulation of a different mechanism involving alternate pathways is probably required to explain the data obtained when p-nitrophenyl phosphate was the substrate.

Inositol phosphatase activity of theEscherichia coli agp-encoded acid glucose-1-phosphatase

2002 ◽  
Vol 48 (9) ◽  
pp. 801-809 ◽  
Author(s):  
Michael A Cottrill ◽  
Serguei P Golovan ◽  
John P Phillips ◽  
Cecil W Forsberg
Keyword(s):  
Escherichia Coli ◽  
Hplc Analysis ◽  
Inositol Phosphate ◽  
Gene Library ◽  
Inositol Phosphatase ◽  
Ph Values ◽  
Hydrolysis Products ◽  
Nitrophenyl Phosphate ◽  
Phosphate Hydrolysis ◽  
Hydrolysis Of

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55°C, while InsP6and p-nitrophenyl phosphate were hydrolyzed maximally at 60°C. The Agp exhibited Kmvalues of 0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5, D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase. Key words: phosphatase, phytate, bacteria, inositol phosphate, phytase.

scholarly journals Eukaryotic Cell Uptake of Heparin-Coated Microspheres: a Model of Host Cell Invasion by Chlamydia trachomatis

2000 ◽  
Vol 68 (3) ◽  
pp. 1080-1085 ◽  
Author(s):  
Richard S. Stephens ◽  
Farah S. Fawaz ◽  
Kathleen A. Kennedy ◽  
Kelly Koshiyama ◽  
Barbara Nichols ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Cell Invasion ◽  
Eukaryotic Cell ◽  
Cell Uptake ◽  
Eukaryotic Cells ◽  
Host Cell Invasion ◽  
Polystyrene Microspheres ◽  
Phagocytic Cells ◽  
Host Proteins ◽  
Lysosomal Compartment

ABSTRACT Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads andChlamydia trachomatis enter eukaryotic cells by similar pathways.

scholarly journals Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

2006 ◽  
Vol 128 (45) ◽  
pp. 14606-14611 ◽  
Author(s):  
Shuhei Imoto ◽  
Jennifer N. Patro ◽  
Yu Lin Jiang ◽  
Natsuhisa Oka ◽  
Marc M. Greenberg
Keyword(s):  
Dna Polymerase ◽  
Nucleoside Triphosphates ◽  
Phosphate Hydrolysis ◽  
Hydrolysis Of

Fine Structural Localization of Acyltransferases Involved in Lipid Synthesis in Intestinal Mucosa.

1970 ◽  
Vol 28 ◽  
pp. 54-55
Author(s):  
R. J. Barrnett ◽  
J. A. Higgins
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Mucosal Cell ◽  
Structural Localization ◽  
Morphological Studies ◽  
Mucosal Cells ◽  
Initial Appearance ◽  
Sh Group ◽  
Hydrolysis Of ◽  
Intestinal Mucosal Cells

The main products of intestinal hydrolysis of dietary triglycerides are free fatty acids and monoglycerides. These form micelles from which the lipids are absorbed across the mucosal cell brush border. Biochemical studies have indicated that intestinal mucosal cells possess a triglyceride synthesising system, which uses monoglyceride directly as an acylacceptor as well as the system found in other tissues in which alphaglycerophosphate is the acylacceptor. The former pathway is used preferentially for the resynthesis of triglyceride from absorbed lipid, while the latter is used mainly for phospholipid synthesis. Both lipids are incorporated into chylomicrons. Morphological studies have shown that during fat absorption there is an initial appearance of fat droplets within the cisternae of the smooth endoplasmic reticulum and that these subsequently accumulate in the golgi elements from which they are released at the lateral borders of the cell as chylomicrons.We have recently developed several methods for the fine structural localization of acyltransferases dependent on the precipitation, in an electron dense form, of CoA released during the transfer of the acyl group to an acceptor, and have now applied these methods to a study of the fine structural localization of the enzymes involved in chylomicron lipid biosynthesis. These methods are based on the reduction of ferricyanide ions by the free SH group of CoA.

Lattice imaging and pore structure of β ferric oxyhydroxide

1978 ◽  
Vol 36 (1) ◽  
pp. 264-265
Author(s):  
T. Baird ◽  
J.R. Fryer ◽  
S.T. Galbraith
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Bulk Material ◽  
Model Structure ◽  
Bulk Crystals ◽  
Lattice Imaging ◽  
Thin Sections ◽  
Ferric Oxyhydroxide ◽  
Resolution Electron ◽  
Hydrolysis Of

Introduction Previously we had suggested (l) that the striations observed in the pod shaped crystals of β FeOOH were an artefact of imaging in the electron microscope. Contrary to this adsorption measurements on bulk material had indicated the presence of some porosity and Gallagher (2) had proposed a model structure - based on the hollandite structure - showing the hollandite rods forming the sides of 30Å pores running the length of the crystal. Low resolution electron microscopy by Watson (3) on sectioned crystals embedded in methylmethacrylate had tended to support the existence of such pores.We have applied modern high resolution techniques to the bulk crystals and thin sections of them without confirming these earlier postulatesExperimental β FeOOH was prepared by room temperature hydrolysis of 0.01M solutions of FeCl3.6H2O, The precipitate was washed, dried in air, and embedded in Scandiplast resin. The sections were out on an LKB III Ultramicrotome to a thickness of about 500Å.

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