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W W Chan

Publications and source records attributed to W W Chan.

At least 19 recordsLinked to original sources
PubMed1992

PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells.

In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRH-stimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10(-6) M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary cells with TRH (10(-7) M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin. sn-1,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent response to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C.

Animals↗
PubMed1979

Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent.

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.

Adenosine Triphosphate↗
PubMed1978

Stabilization of the relaxed state of aspartate transcarbamoylase by modification with a bifunctional reagent.

Native aspartate transcarbamoylase from Escherichia coli was modified with the bifunctional reagent tartaryl diazide in the presence of the substrate carbamoyl phosphate and the substrate analog succinate. The product had the same sedimentation coefficient as the native enzyme but showed a marked increase in affinity for the substrate aspartate with a hyperbolic saturation curve. The Michaelis constant for aspartate (7.4 mM) is similar to that estimated for the relaxed state of the enzyme. The high substrate affinity was not produced if modification was conducted in the absence of substrate analogs or with a monofunctional reagent. The modified enzyme was also desensitized towards the allosteric effectors ATP and CTP. It appears to represent a stabilized relaxed state whose conversion to the taut state is presumably prevented by cross-linking.

Aspartate Carbamoyltransferase↗
PubMed1978

On the mechanism of assembly of the aspartate transcarbamoylase from Escherichia coli.

The mechanism of subunit assembly of aspartate transcarbamoylase from Escherichia coli was studied by following the kinetics of reassociation. The isolated trimetric catalytic subunit (c3) and dimeric regulatory subunit (r2) were mixed together and formation of the dodecameric native enzyme (c6r6) was monitored by measuring changes in activity. Under appropriate conditions the reassociation was second order with respect to the c3 concentration and the effects of varying r2 concentration on the second-order rate constant were examined. An optimum R2 concentration of about 0.07 micrometer was observed. A scheme of the assembly pathways is proposed and is based on the reversible formation of c3r2n (n = 0, 1, 2 or 3) as intermediates. Various combinations of two such c3r2n species are considered as possible rate-limiting steps. This model yields an expression which relates the experimentally determined (overall) second-order rate constant to the equilibrium constant (Kd) governing the formation of c3r2n, the r2 concentration, and four coefficients which reflect the contribution of different types of assembly processes. Using previously determined values of Kd, the above expression for each r2 concentration reduces to a linear equation with four unknowns. The experimental data were subjected to multiple linear-regression analysis and values for the four coefficients were found which gave an excellent fit. Our results show that reassociation of the subunits is a fast bimolecular reaction with rate constants in excess of 10(6) M-1 s-1. Our analysis also suggests that interactions involving a total of more than three r2 subunits (e.g. the combination of c3r2 with c3r6) might contribute significantly to the overall assembly. The influence of various ligands on the reassociation rate profile was also studied. All ligands examined were partially inhibitory to the formation of native enzyme. The effects of substrates were similar to those of CTP whereas the effects of ATP were substantially different. These observations can be readily interpreted by postulating different conformational changes induced by the ligands. These changes should alter the relative orientation of the subunit contacts which must be formed in the reassociation process. The interpretation is consistent with our previous model of the allosteric mechanism.

Aspartate Carbamoyltransferase↗
PubMed1978

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe.

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

Allosteric Regulation↗
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↗