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Biomedical subjects

C Kent

Publications and source records attributed to C Kent.

At least 109 records · Page 6Linked to original sources
PubMed1993

Expression, purification, and characterization of CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.

Bacillus subtilis contains the gene for CTP:glycerol-3-phosphate cytidylyltransferase, which is involved in biosynthesis of the major teichoic acid of the B. subtilis cell wall. When this gene was expressed in Escherichia coli under the control of the T7 promoter, the glycerol-3-phosphate cytidylyltransferase accumulated to a level of about 15% of cellular protein. The expressed glycerol-3-phosphate cytidylyltransferase was purified to homogeneity by ion-exchange chromatography, gel filtration, and affinity chromatography on blue Sepharose. Approximately 47 mg of pure enzyme was obtained from a 660-ml culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the subunit molecular weight of the purified enzyme was about 15,000. The molecular weight of the native enzyme was found to be 30,900 by gel filtration analysis, suggesting that the native enzyme is a homodimer. The pH optimum was very broad, from 6.5 to 9.5, and the enzyme was stable at alkaline conditions. A divalent cation, either Co2+, Mg2+, Mn2+, or Fe2+, was required for enzyme activity. Km values for CTP and glycerol 3-phosphate were 3.85 and 3.23 mM, respectively, and the Vmax was 185 units/mg of protein. Initial rate studies and product inhibition patterns indicated that the enzyme catalyzes the reaction by means of a rapid eqilibrium random order mechanism. The availability of large amounts of glycerol-3-phosphate cytidylyltransferase will facilitate enzymological and structural studies on this model cytidylyltransferase.

Amino Acid Sequence↗
PubMed1993

Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization.

The mechanism by which oleate induced the translocation of CTP:phosphocholine cytidylyltransferase from the soluble to particulate fraction was studied in HeLa cells. Addition of 0.5 mM oleate caused a 10-fold stimulation of [methyl-3H]choline incorporation into phosphatidylcholine. The stimulation of phosphatidylcholine synthesis was correlated with a decrease in phosphocholine and an increase in CDP-choline, indicating that cytidylyltransferase is regulatory under these conditions. The stimulation of cytidylyltransferase activity upon oleate treatment was concomitant with the translocation of this enzyme from the soluble to particulate fraction. Immunoblot analysis revealed the soluble form as multiple slowly migrating bands, and the particulate form as a faster migrating, single band. The slowly migrating form could be converted to the fast migrating form by phosphatase treatment, suggesting that the mobility difference was due to phosphorylation. In vivo 32P labeling showed that the soluble cytidylyltransferase was highly phosphorylated and the particulate form was much less phosphorylated. The kinetics of the translocation showed that the conversion of the majority of cytidylyltransferase from the soluble to the particulate form occurred within about 15 min following addition of oleate and that the translocation showed a good correlation with dephosphorylation. In contrast, the relocation of cytidylyltransferase from the particulate to the soluble fraction after the removal of oleate occurred within 1 min, and extensive phosphorylation was not required for cytidylyltransferase to leave the membrane. The relocated, soluble cytidylyltransferase was phosphorylated at a much slower rate than that at which the enzyme left the membrane. Immunolocalization showed that the cytidylyltransferase translocated to the nuclear envelope instead of the endoplasmic reticulum. These results indicate that activation of cytidylyltransferase by dephosphorylation and translocation to the nuclear envelope may be a general mechanism occurring in many cell types. Moreover, in HeLa cells, multiple mechanism exist for regulating cytidylyltransferase activity.

Biological Transport↗
PubMed1993

Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase.

The soluble form of CTP:phosphocholine cytidylyltransferase, which has previously been assumed to be cytosolic, has been localized to the nucleus of several cell types. Indirect immunofluorescence microscopy indicated a nuclear location in HepG2, NIH-3T3, and L-cells. A comparison of the fluorescence pattern of wild-type CHO cells with a cytidylyltransferase-deficient mutant provided genetic evidence that cytidylyltransferase is nuclear in CHO cells. The enzyme is also predominantly nuclear in rat liver, as revealed by staining frozen sections of that tissue. When L-cells were fractionated by enucleation, over 95% of cytidylyltransferase activity was found in the nuclear fraction, providing biochemical evidence for a nuclear location in these cells. In light of the demonstration that the membrane-bound cytidylyltransferase in CHO cells is associated with the nuclear envelope (Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692), these results suggest that this enzyme is predominantly an intranuclear enzyme.

3T3 Cells↗