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FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly.
- Source :
-
Cell motility and the cytoskeleton [Cell Motil Cytoskeleton] 1998; Vol. 40 (1), pp. 71-86. - Publication Year :
- 1998
-
Abstract
- We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.
- Subjects :
- Actin Cytoskeleton physiology
Animals
Azotobacter vinelandii genetics
Bacterial Proteins genetics
Bacterial Proteins physiology
Chemical Fractionation
Escherichia coli genetics
GTP Phosphohydrolases genetics
GTP Phosphohydrolases physiology
Gene Expression
Gram-Negative Anaerobic Bacteria genetics
Guanosine Triphosphate metabolism
Hydrolysis
Potassium
Rabbits
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Recombinant Fusion Proteins physiology
Sodium
Temperature
Azotobacter vinelandii enzymology
Bacterial Proteins metabolism
Cytoskeletal Proteins
Escherichia coli enzymology
GTP Phosphohydrolases metabolism
GTP-Binding Proteins metabolism
Gram-Negative Anaerobic Bacteria enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0886-1544
- Volume :
- 40
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Cell motility and the cytoskeleton
- Publication Type :
- Academic Journal
- Accession number :
- 9605973
- Full Text :
- https://doi.org/10.1002/(SICI)1097-0169(1998)40:1<71::AID-CM7>3.0.CO;2-I