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I Gibbons

Publications and source records attributed to I Gibbons.

29 records · Page 2Linked to original sources
PubMed1976

A method for the separation of hybrids of chromatographically identical oligomeric proteins. Use of 3,4,5,6-tetrahydrophthaloyl groups as a reversible "chromatographic handle".

Hybridization experiments with variants of an oligomeric protein often provide important information regarding subunit structure, function, and interactions. In some systems, however, the variants are so similar electrophoretically and chromatographically that purification of individual hybrids is not feasible. Therefore a method was developed for preparing hybrids by using 3,4,5,6-tetrahydrophthalic anhydride as a reversible acylating agent for protein amino groups. The technique involved acylating about 30% of the amino groups at pH 8 to give a derivative with a markedly altered net charge, formation of the hybrid set with unmodified and modified species, separation of the individual components by ion-exchange chromatography, and finally removal of the tetrahydrophthaloyl groups from the desired hybrid by incubation for about 1 day at pH 6 and room temperature. Experiments with model compounds and two enzymes showed that the anhydride was sepcific for amino groups. The extent of modification of proteins was measured by the spectral change at 250 nm, the loss of free amino groups, and the change in electrophoretic mobility of the polypeptide chains in polyacrylamide gels containing 8 M urea. Deacylation of modified, inactive aldolase and the catalytic subunit of aspartate transcarbamylase led to the restoration of the enzyme activity and electrophoretic mobility of the unmodified proteins. Both intra- and inter-subunit hybrids of aspartate transcarbamylase were prepared and isolated by using the tetrahydrophthaloyl groups as a reversible "chromatographic handle". Prior to deacylation the inter-subunit hybrid containing one acylated and one native catalytic subunit (and negative regulatory sub-units) exhibited no homotropic cooperativity and after deacylation the characteristic allosteric properties of the enzyme were regained. Similarly the ligand-promoted conformational changes associated with the allosteric transition were resotred upon deacylation of the intra-subunit hybrid containing one acylated and two native chains in each catalytic subunit. Criteria are described which must be satisfied if a reversible "chromatographic handle" is to be effective in hybridization experiments and it is shown that, despite some heterogeneity in its reaction with protein amino groups, 3,4,5,6-tetrahydrophthalic anhydride shows considerable promise for studies of oligomeric proteins.

Binding Sites↗
PubMed1975

Quaternary constraint in hybrid of aspartate transcarbamylase containing wild-type and mutant catalytic subunits.

Unusual quaternary constraint in the regulatory enzyme, aspartate transcarbamylase (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, was demonstrated with a hybrid composed of one inactive "catalytic" subunit from a mutant strain and one active catalytic subunit and three regulatory subunits from the wild-type strain. The hybrid had a high affinity for three molecules of the bi-substrat analog, N-(phosphonacetyl)-L-aspartate, compared to the six strong binding sites in the wild-type enzyme and none in the mutant. However, the Vmax of the hybrid was only about 25% that of the wild-type enzyme. In addition, the hybrid exhibited a very low apparent affinity for the substrate, aspartate [Michaelis constant (Km) about 90 mM], as compared to the wild-type enzyme (apparent Km of 7 mM). No homotropic effect was observed for the hybrid in the absence of nucleotides as contrasted to the cooperativity of the wild-type enzyme; also, large changes in the Vmax of the hybrid were caused by the addition of the nucleotide effectors, CTP and ATP, which do not affect the Vmax of the wild-type, but influence only the cooperativity and the apparent Km. Although the hybrid undergoes a ligand-promoted conformational change analogous to that of the wild-type enzyme, this transition required a 20-fold higher concentration of the substrate analog, succinate. It appears that the "paralysis" of the wild-type catalytic subunit in the hybrid can be attributed to subunit interactions which constrain the molecule in a low-affinity state.

Allosteric Regulation↗
PubMed1974

Kinetic and molecular properties of citraconyl-aldolase. The reversible denaturation and hybridization of the native and modified enzymes.

1. The preparation of enzymically active N-citraconyl derivatives of fructose diphosphate aldolase from rabbit muscle is described. Reaction is restricted to amino groups and the derivatives are not very heterogeneous with respect to the number of substituents. 2. Linear double-reciprocal plots of enzyme velocity against substrate concentration are found up to about 15% blocking of amino groups. With more than 15% blocking, there is a marked downward curvature in the double-reciprocal plots at high substrate concentrations. 3. Over the range 0-25% blocking of amino groups the apparent V(max.) for fructose diphosphate falls to 10% that of the native enzyme, and the apparent K(m) rises from 1 to 400mum. 4. Various pieces of evidence suggest that citraconyl-aldolase is slightly distorted in structure compared with the native enzyme. However, the kinetic properties and tetrameric structure of citraconyl-aldolase can be completely recovered after denaturation in 4m-guanidine hydrochloride. 5. After removal of the citraconyl groups in acid conditions the kinetic and molecular properties of native enzyme are restored. 6. Hybrid forms of aldolase can be constructed containing native and citraconylated subunits and the suitability of these derivatives for the study of subunit interactions in the enzyme is discussed. 7. The kinetic properties of hybridized aldolase containing native and citraconylated subunits are not exactly those predicted from the kinetic properties of the two parental forms. This result is interpreted in terms of conformational changes induced in the native and modified subunits when both are present in a hybrid molecule, evidently as a result of interactions in the tetramer.

Acylation↗
PubMed1974

Subunit interactions in hybrids of native, carboxypeptidase-treated and citraconylated rabbit muscle aldolase.

1. The kinetic properties of hybrids of native (or carboxypeptidase-treated) and citraconylated rabbit muscle aldolase are compared with those of equivalent mixtures of the parental enzymes. 2. In the hybrids, the native subunits function slightly less well than in the homotetramer, but the citraconylated subunits have enhanced activity. 3. Subunits of carboxypeptidase-treated aldolase behave essentially as expected in a hybrid environment, but the citraconylated subunits do not show the same enhancement of activity found in the hybrids of native and citraconylated enzyme. The apparent affinity for fructose 1,6-diphosphate of the citraconylated subunits in hybrids of carboxypeptidase-treated and citraconylated aldolase is increased. 4. These results are interpreted in terms of a substrate-induced conformational difference between native and carboxypeptidase-treated aldolase. 5. This conformational change can take place within a single native subunit in the hybrids and does not require a similar conformational change to occur simultaneously in the other three subunits.

Acylation↗
PubMed1974

Cooperative interactions in aspartate transcarbamoylase. 1. Hybrids composed of native and chemically inactivated catalytic polypeptide chains.

Hybrids of aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate: L-aspartate carbamoyltransferase) from Escherichia coli containing native (active) and pyridoxylated (inactive) catalytic polypeptide chains were constructed by a procedure involving the reversible acylation of amino groups with 3,4,5,6-tetrahydrophthalic anhydride. This technique exploited the charges contributed by the tetrahydrophthaloyl groups as a "chromatographic handle" for separating the various species. Enzyme-like molecules containing one fully active and one inactive catalytic subunit showed cooperative kinetic behavior, considerable inhibition by CTP, and a substantially increased apparent K(m) compared to the native enzyme. Similar properties were observed for an intrasubunit hybrid containing one inactive catalytic polypeptide chain in each subunit. The cooperative inter-and intra-subunit hybrids also exhibited conformational changes similar to those found for the native enzyme upon the addition of stereospecific ligands. These observations, taken together with data for other complexes of catalytic and regulatory subunits, illustrate the importance of the architecture of aspartate transcarbamoylase and the quaternary constraint stemming from the subunit interactions.

Aspartate Carbamoyltransferase↗
PubMed1970

The reaction of aldolase with 2-methylmaleic anhydride.

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.

Amino Acids↗