Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

Rolton, Hilary A.; Keir, Hamish M.

doi:10.1042/bj1410211

Cited by 9 publications

(16 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Part of the deoxycytidine is phosphorylated to dCTP, and a significant portion of the CTP is synthesised from the exogenous deoxycytidine (Table 2). Deamination of dCMP to dUMP has been demonstrated in many eukaryotes [28], but we were not able to measure significant amounts of this enzyme in cell-free extracts by using the method of Maley and Maley [29]. However, we can not exclude the possible function of this enzyme ii?…”

Section: Discussionmentioning

confidence: 96%

Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum

Fink

Nygaard

1978

European Journal of Biochemistry

View full text Add to dashboard Cite

If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to ['4C]02, particularly in the case of [2-'4C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-'4C]thymidine is not associated with DNA.The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases.Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic.The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.Neither pyrimidine bases nor the corresponding nucleosides are obligatory intermediates in the de n o w biosynthesis of nucleotides, rather they are breakdown products of nucleic acids. To what extent Physarum poljwphalum can reutilize breakdown products of nucleic acids is not known.It is probable that Physarum in its natural environment metabolises the nucleic acids of the bacteria and fungi which serve as food source [l]. It is also known that Physarum during differentiation breaks down its own RNA and DNA [2,3] and that Physarum possesses several DNA and RNA-degrading enzymes [4,51.Although plasmodia of Physarum do not require the addition of pyrimidine compounds to the culture medium, these compounds might very well be taken up and converted to nucleotides. In studies of RNA and DNA synthesis in Physurum, radioactively labelled uridine and thymidine have been widely used to label the nucleic acid [6,7].Enzymes. Uridine kinase (EC 2.7.1.48); thymidine kinase (EC 2.7.1.21); deoxycytidine kinase (EC 2.7.1.74); uridine nucleosidase (hydrolase) (EC 3.2.2.3); cytidine dedminase (EC 3.5.4.5); UMP pyrophosphorylase (EC 2.4.2.9).In order to elucidate this, we have in the present investigation determined the extent to which pyrimidine nucleosides and bases can be taken up and metabolised by microplasmodia of Physarum. Our data show (a) that nucleosides in contrast to pyrimidine bases are readily taken up and converted into nucleotides and acid-insoluble material, (b) that there is a substantial catabolism of nucleosides, and (c) that incorporation of radioactive deoxyribonucleosides into total acid-insoluble material is not a reliable method for quantitative determination of DNA.Some of the enzyme reactions involved in the metabolism of nucle...

show abstract

Section: Discussionmentioning

confidence: 96%

Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum

Fink

Nygaard

1978

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…At this stage they were either harvested for immediate enzyme extraction, or incubated for a further 3-4 days in ETCo.5 medium before being harvested for enzyme extraction. During maintenance in ETCo.5 medium, metabolic activities of the cells decline to a basal level after 3-4 days (Burk, 1970;Rolton & Keir, 1974). The cells are then said to be quiescent.…”

Section: Methodsmentioning

confidence: 99%

Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid

Craig

Costello

Keir

1975

Biochemical Journal

View full text Add to dashboard Cite

BHK-21/C13 cells were grown in culture under conditions that provided exponentially growing cells and quiescent cells, by modifying the concentration of serum in the growth medium. The high-molecular-weight DNA polymerase (DNA polymerase I) from exponentially growing cells accounted for 90% of the total polymerase activity; the low-molecular-weight DNA polymerase (DNA polymerase II) accounted for the remaining 10%. In quiescent cells, DNA polymerase I contributed only 39% of the total polymerase activity and DNA polymerase II 61%. The total amount of DNA polymerase I in exponentially growing cells was 11.3-fold greater than that in quiescent cells, whereas the amount of DNA polymerase II appeared to be relatively independent of the physiological state of the cells. In an extension of these experiments, cells in a quiescent state (Go cells) were stimulated by the 'serum-step-up' method of Burk (1970) to grow and to enter a synchronous wave of DNA synthesis (S-phase cells), 87% of the cells synthesizing DNA at 20 h after the 'serum-step-up'. During the synchrony experiment, the total cytoplasmic and total nuclear DNA polymerase activities each increased about 4-fold in parallel with the increase in the rate of DNA synthesis. Cytoplasmic polymerase activity was always greater than nuclear polymerase activity. The increases observed were maximal at 20 h after 'serum step-up'. By 26 h, there was a decrease in enzyme activity (8% for cytoplasmic polymerase and 16% for nuclear polymerase, both relative to the maximum at 20 h), but the rate of DNA synthesis had declined by 37% relative to the maximum at 20 h. In Go cells, DNA polymerase II (mol.wt. 46000 +/- 4000) was the predominant species, there being twice as much of it as of the total DNA polymerase I. In these cells there was little DNA polymerase IC and ID; the amounts of IA (mol.wt. 900 times 10(3)-1100 times 10(3)) and IB (mol.wt. 460 times 10(3)-560 times 10(3)) were about equal but small.

show abstract

“…Cell culture. BHK-21/C13 cells were grown as described by Rolton & Keir (1974). ETC10 is Eagle's (modified) medium containing 10% tryptose phosphate broth and 10% (v/v) calf serum;…”

Section: Methodsmentioning

confidence: 99%

“…Preparation ofcell extracts for enzyme purification. Cells were disrupted and extracted as described by Rolton & Keir (1974) except that homogenization was carried out with Brij 35 (0.5%, w/v) in the buffer to ensure efficient cell breakage. The detergent did not affect the ensuing enzyme assays.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Deoxycytidylate deaminase. Evidence for a new enzyme in cells infected by the virus of herpes simplex

Rolton

Keir

1974

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

The activities of dCMP deaminase and DNA polymerase I increased twofold and fivefold in BHK-21/C13 cells after infection by the virus of herpes simplex. The increases were greatly diminished, and under certain conditions prevented, by inclusion of actinomycin D or cycloheximide in the cell-virus system during the infective cycle. The dCMP deaminase purified from infected cells harvested 8h after infection differed from the deaminase purified from non-infected cells inasmuch as (a) it was more resistant to heating at 37 degrees C; (b) the substrate (dCMP) concentration at half-maximum velocity was lower; (c) maximum activation was achieved by a lower concentration of dCTP; (d) it was more resistant to inhibition by dTTP; and (e) it behaved differently when assayed in the presence of a herpes-virus-specific antiserum. The DNA polymerase activity in the infected cells was markedly decreased in the presence of the herpes-virus-specific antiserum.

show abstract

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

Cited by 9 publications

References 30 publications

Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum

Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum

Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid

Deoxycytidylate deaminase. Evidence for a new enzyme in cells infected by the virus of herpes simplex

Contact Info

Product

Resources

About