Conformational Changes in Aspartate Transcarbamylase

McClintock, David K.; Markus, Gabor

doi:10.1016/s0021-9258(18)93350-8

Cited by 71 publications

(14 citation statements)

References 14 publications

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“…After digestion for 20min, the effects of 2mM-ATP and 0.2mM-CTP on the reaction are negligible, in agreement with the results of McClintock & Markus (1968), and the specific activity of the solution is higher than at zero time (Fig. 1).…”

Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasesupporting

confidence: 88%

“…It appeared that trypsin preferentially attacked the regulatory subunits of the enzyme. These and other studies (McClintock & Markus, 1968;Markus et al, 1971) indicated that an investigation of the tryptic digestion of native aspartate transcarbamoylase would be of interest with regard to the structure of the enzyme. Two catalytically active proteins differing from the native enzyme have been isolated from limited tryptic digests of the native enzyme, and some properties of these species are reported.…”

mentioning

confidence: 88%

“…Native enzyme (5.5mg/ml) was incubated at 28'C with trypsin (0.4mg/ml) in 0.05 M-sodium borate buffer, pH8.5, containing lOmM-aspartate (McClintock & Markus, 1968), for various time-periods. The reaction was stopped by the addition of di-isopropyl phosphorofluoridate to give a final concentration of 0.1 mM.…”

Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasementioning

confidence: 99%

“…Velocities are expressed as ,mol of carbamoylaspartate formed/min per 0.04,1l of digestion mixture. The percentage of protein digested (o) was determined by measuring the formation of HCl03-soluble peptides, which were determined by the method of McClintock & Markus (1968). 1973 34.…”

Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasementioning

confidence: 99%

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Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin

Heyde

Nagabhushanam

Venkataraman

1973

Biochemical Journal

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1. The time-course of tryptic hydrolysis of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) was followed by activity measurements in the presence and absence of allosteric effectors, and by polyacrylamide-gel electrophoresis. 2. Two proteins with enzyme activity are formed in this way from native enzyme, and the isolation and some properties of these species are reported. The larger protein (10.6S) resembles native enzyme in that it contains regulatory subunits and is sensitive to allosteric effectors, as well as in a more detailed kinetic investigation. It appears from the time-course of tryptic digestion to be an intermediate in the formation of a catalytic subunit (5.5S) which is similar to, but not identical with, the catalytic subunit produced by mercurial treatment of the native enzyme. 3. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of the different enzyme forms demonstrates that trypsin can hydrolyse bonds in the catalytic polypeptide chains as well as completely remove the regulatory polypeptide chains. 4. Both preparations of catalytic subunit can recombine with regulatory subunit to form enzymes which resemble the native enzyme in being activated by ATP, although they do not appear to be inhibited by CTP. 5. This study is consistent with the models of the enzyme that propose that the catalytic subunits are held together in the native enzyme by three pairs of regulatory polypeptide chains.

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Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasesupporting

confidence: 88%

mentioning

confidence: 88%

Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasementioning

confidence: 99%

Section: Tryptic Hydrolysis Of Aspartate Transcarbamoylasementioning

confidence: 99%

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Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin

Heyde

Nagabhushanam

Venkataraman

1973

Biochemical Journal

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“…A large body of evidence indicates that the binding of nucleotides to r2 alters both tertiary structure and subunit interactions [cf. McClintock & Markus (1968), Cohlberg et al (1972), Hensley & Schachman, (1979), Lennick & Allewell (1981), and Honzatko et al (1982)], with CTP, which binds more tightly than ATP, generally having a larger effect. The dissociation heats necessarily include any contributions from structural changes in the protein.…”

Section: Discussionmentioning

confidence: 99%

High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase

Edge¹,

Allewell²,

Sturtevant³

1985

Biochemistry

141

112

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The thermal denaturation of the catalytic (c3) and regulatory (r2) subunits of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of highly sensitive differential scanning calorimetry. The denaturation of both types of subunit is irreversible as judged by the facts that the proteins coagulate when heated and that no endotherm is observed when previously scanned protein is rescanned. Despite this apparent irreversibility, there is empirical justification for analyzing the calorimetric data in terms of equilibrium thermodynamics as embodied in the van't Hoff equation. The observed curves of excess apparent specific heat vs. temperature are asymmetric and can be expressed within experimental uncertainty as the sums of sequential two-state steps, a minimum of two steps being required for r2 and three for c3. As previously reported [Vickers, K. P., Donovan, J. W., & Schachman, H. K. (1978) J. Biol. Chem. 253, 8493-8498], the addition of the effectors ATP and CTP raises the denaturation temperature of r2 and lowers that of c3 while the addition of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate raises the denaturation temperature of c3 and lowers that of r2. These effects vary with ligand concentration in the manner expected from the van't Hoff equation, indicating that they are simply manifestations of Le Chatelier's principle rather than being due to "stabilization" or "destabilization" of the proteins. The denaturational enthalpy is increased in those cases of ligand binding in which the denaturation temperature is increased, because of the contribution from the enthalpy of dissociation of the ligand.

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Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

et al. 2000

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Conformational Changes in Aspartate Transcarbamylase

Cited by 71 publications

References 14 publications

Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin

Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin

High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase

Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

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