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Biomedical subjects

W W Chan

Publications and source records attributed to W W Chan.

At least 91 records · Page 5Linked to original sources
PubMed1977

Dissociation of phosphohistone phosphatases from canine heart.

Phosphohistone phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of canine heart extract has been separated by DEAE-cellulose chromatography into 4 molecular forms, namely phosphatases A (Mr = 156 000), B (Mr = 161 000), C (Mr = 95 600) and U (Mr = 61 000). ATP inhibited phosphatase A, stimulated phosphatase B and did not significantly affect phosphatase C activity. Phosphatase U requires Mn2+ for activity, under which condition ATP is inhibitory. Phosphatases A, B and C, but not phosphatase U, were dissociated by ethanol into catalytic subunits that were inhibited by ATP, insensitive to Mn2+, and had a common molecular weight of 34 800 (phosphatase S). The dissociation was accompanied by an increase of enzymic activity. Chromatography of the ethanol-treated 55% (NH4)2SO4 fraction of canine heart extract on DEAE-cellulose demonstrated that the multiple forms of phosphohistone phosphatase could be reduced to two forms: phosphatase U and phosphatase S, which may represent two basic constituents of the multiple forms of phosphohistone phosphatase in canine heart.

Adenosine Triphosphate↗
PubMed1976

Effects of subunit interactions on the activity of lactate dehydrogenase studied in immobilized enzyme systems.

Rabbit muscle lactate dehydrogenase (LDH) was coupled to Sepharose in such a way that each molecule is expected to be attached via only one subunit. Dissociation of the bound active enzyme by several methods all yielded immobilized subunit derivatives which were inactive. These derivatives were capable of regenerating activity by interacting specifically with subunits in solution formed transiently during renaturation. This ability to peck up soluble subunits is lost fairly rapidly upon storage of the immobilized subunits. Similarly, LDH subunits attached to Sepharose via disulfide bonds were found to be inactive. When these subunits were detached from the matrix by mild reduction with mercaptoethanol, activity was regenerated. The kinetics of this reactivation process suggests that reassociation is required for appearance of activity. All these results can be interpreted as showing that subunit interactions are essential for LDH activity.

Animals↗
PubMed1976

Further studies on aspartate transcarbamoylase: molecular weight of the c3r6 complex and analysis of succinate inhibition in the native enzyme.

The complex which is formed when excess regulatory subunits (r2) of aspartate transcarbamoylase (EC2.1.3.2) are added to a dilute solution of the catalytic subunit (c3) has been studied by gel-filtration on Sephadex G-200. The elution volume indicates a Stokes' radius of between 5.42 and 5.92 nm, depending on the method of calculation. Using the sedimentation coefficient of 7.7S previously determined, the molecular weight is estimated to be close to 200 000, in support of the c3r6 structure proposed earlier for the complex. The calculated frictional coefficient indicates abnormal hydrodynamic properties which are probably due to unusual structure characteristics. The pattern of succinate inhibition of native aspartate transcarbamoylase has also been analyzed. At low concentrations, succinate activates the enzyme, presumably by converting it from the taut state to the relaxed (r) state. Further increase in the succinate concentration leads to competitive inhibition of the R state. Using a novel procedure for analysis of the data, the Michaelis constant for aspartate of the R state has been estimated to be about 7mM. This value is close to the Km of c3r6 for aspartate, measured under identical conditions. The result therefore provides further evidence suggesting that the c3r6 complex resembles the R state of the native enzyme.

Aspartate Carbamoyltransferase↗
PubMed1976

Electrical off response of cat esophageal smooth muscle: an analog simulation.

This investigation was performed to define characteristics and to design an electrical model of the slow membrane depolarization, or "electrical off response," that follows stimulation of cat circular esophageal smooth muscle. The electrical off response showed the following characteristics: 1) the response was preceded by hyperpolarization of the muscle membrane; 2) the amplitude of the off-response depolarization increased directly with increasing amplitude of the preceding hyperpolarization; 3) the membrane potential showed varying degrees of oscillation around the resting point; 4) the oscillation period was constant (4-5 s) and was double the time from the point of maximum hyperpolarization to the peak of the electrical off response. These characteristics suggested that the electrical off response could be represented by a linear second-order differential equation with variable damping, and an analog simulation was constructed. By varying the damping ratio and/or the natural frequency of the analog model, the characteristics of the electrical off response of esophageal muscle could be reproduced. The model provides a means to explore the behavior of esophageal smooth muscle contraction under normal and pathological conditions.

Animals↗
PubMed1975

Subunit interactions in aspartate transcarbamylase. The interaction between catalytic and regulatory subunits and the effect of ligands.

The interaction between the catalytic subunit (c3) and the regulatory subunit (r2) of aspartate transcarbamylase from Escherichia coli was studied by measuring the reversible formation of the c3r6 complex as a function of r2 concentration. Conversion to the native enzyme was prevented by using a very low concentration of c2 (40 ng per ml) in the presence of bovine serum albumin. A simple hyperbolic r2 saturation curve was obtained suggesting the presence of only one kind of c:r domain. From the association constant for the formation of c3r6, the free energy of c:r interaction can be estimated to be about -10 Cal per mole. Neither CTP nor ATP appears to affect the strength of c:r interaction in this complex. Succinate in the presence of carbamyl phosphate promotes tighter binding. At higher concentration of c3 and nonsaturating levels of r2, conversion to the native enzyme (c3r6) takes place. This renaturation process is second order with respect to the concentration of c3 and is virtually irreversible. Renaturation is inhibited by saturating levels of r2 and to some extent by both CTP and ATP. The effect of ligands on c:r interactions reported here may have significance in the allosteric mechanism of the native enzyme.

Adenosine Triphosphate↗
PubMed1975

Subunit interactions in aspartate transcarbamylase. A model for the allosteric mechanism.

The conformational changes in aspartate transcarbamylase upon binding of substrates or regulatory ligands and the effects of alterations in the subunit structure on the allosteric interactions are reviewed. The available information including recent results from studies of the c3r6 complex (c denotes the catalytic polypeptide and r, the regulatory polypeptide) is considered in terms of the existing models for the discrepancies between experimental observations and the present models could be resolved by postulating an important role for r:r interactions in the allosteric mechanism. A new model is presented in which an obligatory conformational change upon binding of substrates results in an alteration in the relative orientation of c versus r. As a consequence of symmetry conservation, the r:r domain is shifted to a position of higher potential energy. By favoring one or the other alternative r:r domains, CTP and ATP can respectively enhance and reduce the sigmoidal character of substrate saturation. The model is shown to be consistent with all of the important known properties of the enzyme. Because the heterotropic effects of CTP or ATP are postulated to operate via a mechanism separate from that for the homotropic effects of the substrates, this model accounts satisfactorily for the observation by Kerbiriou and Herve (Kerbiriou, D., and Herve, G. (1973) J. Mol. Biol. 78, 687-702) that homotropic effects can be abolished whereas heterotropic effects are retained in the altered enzyme from Escherichia coli grown in the presence of 2-thiouracil.

Adenosine Triphosphate↗
PubMed1975

Subunit interactions in aspartate transcarbamylase. Characterization of a complex between the catalytic and the regulatory subunits.

The complex formed when excess regulatory subunits (r2) of aspartate transcarbamylase is added to a dilute solution of the catalytic subunit (c3) has been further studied. By stabilizing the complex with saturating levels or r2, it was possible to perform ultracentrifugation in sucrose density gradients. The sedimentation coefficient of the complex (7.7 plus or minus 0.2 S) is intermediate between those of the catalytic subunit (5.8 S) and of the native enzyme (11.7 S). Consideration of the likely hydrodynamic properties of the complex suggests that this sedimentation coefficient may be consistent with the c3r6 structure previously proposed. The formation of c3r6 from c3 and r2 is readily reversible. At nonsaturating levels or r2, conversion to the native enzyme (c3r6) takes place. This conversion is inhibited by high concentrations of r2. The c3r6 complex shows Michaelis-Menten kinetics with a low Km for aspartate and considerable substrate inhibition. The pH activity profile at high aspartate concentrations is almost identical with that of the native enzyme. All of these observations suggest that c3r6 represents the relaxed (R) state of aspartate transcarbamylase. The insensitivity of c3r6 toward CTP or ATP can also be explained by considering c3r6 as a stabilized relaxed state. These properties of c3r6 have significant implications regarding the allosteric mechanism of the native enzyme.

Adenosine Triphosphate↗