ABSTRACT
Recently, a native bacteriohemerythrin (McHr) has been identified in Methylococcus capsulatus (Bath). Both the particulate methane monooxygenase (pMMO) and McHr are over-expressed in cells of this bacterium when this strain of methanotroph is cultured and grown under high copper to biomass conditions. It has been suggested that the role of the McHr is to provide a shuttle to transport dioxygen from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by the pMMO. Indeed, McHr enhances the activity of the pMMO when pMMO-enriched membranes are used to assay the enzyme activity. We find that McHr can dramatically improve the activity of pMMO toward the epoxidation of propylene to propylene oxide. The maximum activity is observed at a pMMO to McHr concentration ratio of 4:1, where we have obtained specific activities of 103.7nmol propylene oxide/min/mg protein and 122.8nmol propylene oxide/min/mg protein at 45°C when the turnover is driven by NADH and duroquinol, respectively. These results are consistent with the suggestion that the bacterium requires McHr to deliver dioxygen to the pMMO in the intra-cytoplasmic membranes to accomplish efficient catalysis of methane oxidation when the enzyme is over-expressed in the cells.
Subject(s)
Bacterial Proteins/pharmacology , Hemerythrin/pharmacology , Methylococcus capsulatus/drug effects , Oxygenases/metabolism , Alkenes/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis/drug effects , Cell Membrane/drug effects , Cell Membrane/enzymology , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/drug effects , Epoxy Compounds/metabolism , Hemerythrin/genetics , Hemerythrin/metabolism , Hydroquinones/pharmacology , Membrane Proteins/metabolism , Methane/metabolism , Methylococcus capsulatus/enzymology , NAD/pharmacology , Oxidation-Reduction/drug effects , Oxygen/metabolism , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , SpectrophotometryABSTRACT
Hemerythrin is a dioxygen-carrying protein whose oxidative/nitrosative stress-related reactivity is lower than that of hemoglobin, which may warrant investigation of hemerythrin as raw material for artificial oxygen carriers ('blood substitutes'). We report here the first biological tests for hemerythrin and its chemical derivatives, comparing their performance with that of a representative competitor, glutaraldehyde-polymerized bovine hemoglobin. Hemerythrin (native or derivatized) exhibits a proliferative effect on human umbilical vein endothelial cell (HUVEC) cultures, as opposed to a slight inhibitory effect of hemoglobin. A similar positive effect is displayed on human lymphocytes by glutaraldehyde-polymerized hemerythrin, but not by native or polyethylene glycol-derivatized hemerythrin.
Subject(s)
Blood Substitutes/pharmacology , Endothelial Cells/physiology , Hemerythrin/pharmacology , Hemoglobins/pharmacology , Leukocytes/physiology , Umbilical Veins/cytology , Adult , Animals , Blood Substitutes/chemistry , Cattle , Cell Line , Cell Survival/drug effects , Cross-Linking Reagents/chemistry , Endothelial Cells/drug effects , Glutaral/chemistry , Hemerythrin/chemistry , Hemoglobins/chemistry , Humans , Leukocytes/drug effects , MaleABSTRACT
OBJECTIVES: To investigate the immune defense of the annelid Nereis diversicolor and the key role of a oxygen-binding protein, the metalloprotein MPII animals were subjected to bacteria infection. METHODS AND RESULTS: Using RACE-PCR, we have cloned the complete cDNA coding for the MPII related to the hemerythrin family in the sand worm Hediste diversicolor. This cDNA (883 pb) codes for a polypeptide of 119 amino acid residues with no signal peptide. Previous works have identified this protein as a cadmium scavenger. We here clearly demonstrated that this protein is also involved in the worm defence towards bacteria growth by its iron scavenger ability. This protein is expressed and produced in a haematopoietic center that floats freely in the coelomic fluid before stored in a particular hemocyte type: the granulocyte type 1. During bacterial challenge, this protein contained in these cells is discharged into the blood stream 3-4 hours after the infection and remains active for approximately 10 hours. This time period blocks progression of the pathogen and its attachment to tissues. CONCLUSION: These results reflect that MPII in conjunction with others partners like lysozyme act as defence molecule for the sand worm.
Subject(s)
Annelida/chemistry , Anti-Bacterial Agents , Hemerythrin/pharmacology , Amino Acid Sequence , Animals , Annelida/immunology , Bacteria/drug effects , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/drug effects , Escherichia coli/growth & development , Granulocytes/metabolism , Hemerythrin/biosynthesis , Hemerythrin/genetics , Immunohistochemistry , In Situ Hybridization , Microbial Sensitivity Tests , Molecular Sequence Data , RNA/biosynthesis , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Our earlier studies showed that rabbit muscle phosphoglucomutase was irreversibly inactivated by exposure to a mixture of vitamin C, FeCl3 and O2. The enzyme lost about 70% of its phosphate (V.V. Desphande and J.G. Joshi, J. Biol. Chem. 260, 754-764, 1985). The present report shows that several other iron proteins can substitute for FeCl3 to a varying degree. The rate of inactivation by FeCl3 greater than ferritin greater than hemoglobin = hemerythrin greater than transferrin = ferridoxin = vitamin C. These iron compounds also produced dephosphoenzyme but did not dephosphorylate ATP, ADP, AMP or phospholipids.
