Heat-induced disaggregation of a multifunctional enzyme complex catalyzing the first steps in pyrimidine biosynthesis in bakers' yeast

1970 ◽  
Vol 48 (2) ◽  
pp. 155-159 ◽  
Author(s):  
Peter F. Lue ◽  
J. G. Kaplan
Keyword(s):  
Feedback Inhibition ◽  
Sucrose Density Gradient ◽  
Enzyme Complex ◽  
Arrhenius Law ◽  
Aspartate Transcarbamylase ◽  
Uridine Triphosphate ◽  
Alternative Hypotheses ◽  
Atcase Activity ◽  
Carbamylphosphate Synthetase ◽  
Duration Of Heating

A highly purified enzyme complex (from bakers' yeast), which possesses carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) activities and a regulatory site for both activities at which feedback inhibition is exerted by UTP, was subjected to heat at 50 °C, and all three of the above parameters were followed. A treatment of 5 min was sufficient to cause disaggregation of the complex (original molecular weight 600 000) and appearance of subunits of approximately one-quarter the size of the unheated material. These subunits possessed only the ATCase activity, which was insensitive to uridine triphosphate; no component possessing CPSase activity was found in the sucrose density gradient. The ATCase was relatively resistant to heat, but there was a rapid and roughly parallel decline in the CPSase and in the sensitivity of the ATCase to inhibition by UTP. However, the residual CPSase activity, at any duration of heating, remained fully sensitive to inhibition by uridine triphosphate. CPSase activity was optimal at temperatures between 25° and 30° whereas the ATCase followed the Arrhenius law to 37 °C. Two alternative hypotheses of the structure of the enzyme complex were put forward to account for these data. Both of these hypotheses require that CPSase activity be a function of the oligomeric aggregate and not of individual subunits.

Aggregation States of a Regulatory Enzyme Complex Catalyzing the Early Steps of Pyrimidine Biosynthesis in Bakers' Yeast

1971 ◽  
Vol 49 (4) ◽  
pp. 403-411 ◽  
Author(s):  
P. F. Lue ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Crude Extract ◽  
Analytical Ultracentrifugation ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase

Aspartate transcarbamylase (ATCase) of bakers' yeast has been purified 78-fold from a crude extract of a derepressed diploid strain; its specific activity was more than 300-fold that of a wild-type crude extract. During the last steps of the purification there was a parallel co-purification of carbamylphosphate synthetase (CPSase), and both activities retained full sensitivity to feedback inhibition by UTP; indeed the sensitivity of the ATCase to UTP increased during the purification doubtless due to discard of a feedback-insensitive ATCase subunit. The two enzyme activities co-eluted from gel filtration on Sepharose 6B together with the feedback site. Analytical ultracentrifugation revealed that the material was not homogeneous, showing two major peaks. Sucrose density gradient centrifugation in the presence of UTP, glutamine, and Mg2+ resulted in co-sedimentation of the two activities and the regulatory site, corresponding to a molecular weight of approximately 800 000 daltons. Omission of UTP from the gradient resulted in disappearance of the heavy peak and appearance of a new one, corresponding to a molecular weight of 380 000 and possessing both activities; the CPSase was still highly sensitive to UTP unlike the ATCase which was only slightly sensitive to retroinhibition. Omission of glutamine and Mg2+ from the sucrose density gradient caused a distinct CPSase peak to trail behind the ATCase; again, the CPSase (molecular weight 250 000) retained full sensitivity to feedback inhibition. This, together with genetic data, supports the view that the ura-2 gene which controls ATCase, CPSase, and the regulatory site is a polycistronic operon, coding for the production of two or three polypeptide chains; the CPSase subunit is inactive unless a regulatory site is present, whereas the ATCase subunit (molecular weight 140 000) is highly active but completely insensitive to feedback inhibition.

scholarly journals Structural and functional relationships of enzyme activities induced by nitrate in barley

1970 ◽  
Vol 119 (4) ◽  
pp. 715-725 ◽  
Author(s):  
John L. Wray ◽  
Philip Filner
Keyword(s):  
Cytochrome C ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Bottom Layer ◽  
Sucrose Density Gradient ◽  
Enzyme Complex ◽  
Cytochrome C Reductase ◽  
Functional Relationships ◽  
Nadh Cytochrome

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH2–nitrate reductase and NADH–cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH–nitrate reductase (8S), one band of FMNH2–nitrate reductase activity (8S) and three bands of NADH–cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH–cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH–cytochrome c reductase band co-sediments with both NADH–nitrate reductase activity and FMNH2–nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH–cytochrome c reductase. 4. FMNH2–nitrate reductase activity is more stable (t½ 12.5min) than either NADH–nitrate reductase activity (t½ 0.5min) or total NADH–cytochrome c reductase activity (t½ 1.5min) at 45°C. 5. NADH–cytochrome c reductase and NADH–nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH2–nitrate reductase activity. 6. Tungstate prevents the formation of NADH–nitrate reductase and FMNH2–nitrate reductase activities, but it causes superinduction of NADH–cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH–cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH–nitrate reductase, FMNH2–nitrate reductase and NADH–cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH–cytochrome c reductase activity.

Effects of salts on the aspartate transcarbamylase of a halophilic eubacterium, Vibrio costicola

1989 ◽  
Vol 67 (9) ◽  
pp. 666-669 ◽  
Author(s):  
Ijeoma Ahonkhai ◽  
Masahiro Kamekura ◽  
Donn J. Kushner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Activity ◽  
Ammonium Sulphate ◽  
Prolonged Storage ◽  
Response Patterns ◽  
Aspartate Transcarbamylase ◽  
Moderately Halophilic ◽  
Low Salt ◽  
Salt Response ◽  
Atcase Activity

The aspartate transcarbamylase (ATCase) in cell-free extracts of the moderately halophilic eubacterium, Vibrio costicola, was stable in 1.5 M NaCl, but not in 0.5 M NaCl on prolonged storage at 4 °C in concentrated extracts. At lower salt concentrations, activity was lost rapidly. ATCase activity was optimal at about 1.5 M NaCl or 1.0 M KCl, although high activity was detected at 0.15 M NaCl. In the presence of 0.03 M aspartate both succinate and maleate inhibited ATCase activity. CTP inhibited the activity of the enzyme at low salt concentrations (0.15 to 0.3 M). Much less inhibition occurred at higher salt concentrations. Precipitating the enzyme with ammonium sulphate resulted in loss of CTP inhibition. The ATCase of V. costicola differs from those of a nonhalophile (Saccharomyces cerevisiae) and an extremely halophilic archaebacterium (Halobacterium cutirubrum) in its salt-response patterns of activity and regulation.Key words: halophilic, aspartate transcarbamylase, Vibrio costicola.

Kinetics and Reaction Mechanism of the Carbamylphosphate Synthetase of a Multienzyme Aggregate from Yeast

1975 ◽  
Vol 53 (6) ◽  
pp. 721-730 ◽  
Author(s):  
David M. Aitken ◽  
Peter F. Lue ◽  
J. Gordin Kaplan
Keyword(s):  
Reaction Mechanism ◽  
Feedback Inhibition ◽  
Enzyme Reaction ◽  
Competitive Inhibitor ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
Nitrogen Donor ◽  
Sequential Addition ◽  
A Site ◽  
Carbamylphosphate Synthetase

We have studied the kinetics and reaction mechanism of the carbamylphosphate synthetase of an enzyme aggregate functioning in the pyrimidine pathway of yeast. Mg–ATP was found to be one of the substrates of the enzyme reaction which was activated by free Mg2+ and inhibited by free ATP. Feedback inhibition by UTP was non-competitive with respect to both glutamine and bicarbonate. Potassium ions were essential for activity and could not be replaced by sodium. Glutamine could be replaced partially by ammonium ions as nitrogen donor. A bicarbonate-dependent cleavage of ATP was shown to take place in the absence of L-glutamine; L-glutamate was a competitive inhibitor of L-glutamine and the enzyme was shown to synthesize ATP when incubated with ADP and carbamyl phosphate. The reaction mechanism was found to involve sequential addition of the substrates bicarbonate and Mg–ATP and release of ADP, followed by addition of the third substrate glutamine. The purine nucleotide XMP had a pronounced activating effect on the enzyme, acting at a site different from that of UTP. Saturating levels of Mg–ATP eliminated this activation.

scholarly journals Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

1986 ◽  
Vol 6 (9) ◽  
pp. 3050-3058 ◽  
Author(s):  
J C Ruiz ◽  
G M Wahl
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Expression Vectors ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
High Concentrations ◽  
Atcase Activity ◽  
Ovary Cell Line

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

scholarly journals Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae.

1982 ◽  
Vol 92 (3) ◽  
pp. 790-794 ◽  
Author(s):  
M Nagy ◽  
J Laporte ◽  
B Penverne ◽  
G Hervé
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Feedback Inhibition ◽  
Nuclear Accumulation ◽  
Lead Phosphate ◽  
Carbamoylphosphate Synthetase ◽  
Aspartate Carbamoyltransferase ◽  
Encoding Gene ◽  
Atcase Activity ◽  
Cytochemical Technique

The cytochemical technique using the in situ precipitation of orthophosphate ions liberated specifically by the aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) reaction indicated that in Saccharomyces cerevisiae this enzyme is confined to the nucleus. This observation is in accordance with the result reported by Bernhardt and Davis (1972), Proc. Natl. Acad. Sci. U. S. A. 69:1868-1872) on Neurospora crassa. The nuclear compartmentation was also observed in a mutant strain lacking proteinase B activity. This finding indicates that this proteinase is not involved in the nuclear accumulation of ATCase, and that the activity observed in the nucleus corresponds to the multifunctional form associated with the uracil path-specific carbamoylphosphate synthetase and sensitive to feedback inhibition by UTP. In a ura2 strain transformed by nonintegrated pFL1 plasmids bearing the URA2-ATCase activity encoding gene, the lead phosphate precipitate was observed predominantly in the cytoplasm. This finding enhances the reliability of the technique used by eliminating the possibility of an artifactual displacement of an originally cytoplasmic reaction product during the preparation of the material for electron microscopy. On the other hand, nuclei isolated under hypoosmotic conditions do not exhibit the ATCase activity that is recovered in the cytosolic fractions after differential centrifugation of the lysate in Percoll gradient. A release of the protein from the nuclei during the lysis step, consistent with its nucleoplasmic localization, is postulated.

The combined effects of temperature and dilution on the activity and feedback inhibition of yeast aspartate transcarbamylase

1969 ◽  
Vol 47 (4) ◽  
pp. 477-479 ◽  
Author(s):  
J. G. Kaplan ◽  
Irmgard Messmer
Keyword(s):  
Low Temperature ◽  
Sharp Increase ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Combined Effects ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase ◽  
Small Loss ◽  
Progressive Loss ◽  
Effects Of Temperature

Dilution of a partially purified preparation of yeast aspartate transcarbamylase caused only a small loss of feedback inhibition and no change in specific activity when the assay was carried out at low temperature (0–10 °C). As the temperature of assay was increased, there was in the case of the dilute preparation a progressive loss of feedback inhibition coupled with a sharp increase in specific activity, reaching a level 400–500% that of the concentrated preparations. The data suggest that this is due to a dissociation into subunits possessing high aspartate transcarbamylase activity but lacking the regulatory site.

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