title: Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils.
abstract: Supernatants prepared from disrupted Coxiella burnetii possess acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M(r) of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 degrees C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 degrees C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-linked glycan residues since its treatment with N-glycosidase F (PNGase F) decreased its M(r) to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phosphocompounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP > 5'-AMP. The ACP also dephosphorylated (32)P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C. burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells.
authors.affiliation: Biology Department, University of New Mexico, Albuquerque 87131-1091, USA.
authors.forename: Y P
authors.initials: YP
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Curley
authors.forename: M
authors.initials: M
authors.lastname: Lopez
authors.forename: M
authors.initials: M
authors.lastname: Chavez
authors.forename: R
authors.initials: R
authors.lastname: Glew
authors.forename: A
authors.initials: A
authors.lastname: Aragon
authors.forename: H
authors.initials: H
authors.lastname: Kumar
authors.forename: O G
authors.initials: OG
authors.lastname: Baca
mesh_terms: D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000963:Antimetabolites / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016997:Coxiella burnetii / Q000201:enzymology*; D017886:Durapatite; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007739:L Cells; D051379:Mice; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D014443:Tyrosine; D014638:Vanadates
chemical_list: D000963:Antimetabolites; D001426:Bacterial Proteins; D014638:Vanadates; D014443:Tyrosine; D017886:Durapatite; D000135:Acid Phosphatase; D017027:Protein Tyrosine Phosphatases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase