Enzyme synthesis in the regulation of hepatic ‘malic’ enzyme activity

Murphy, Gillian; Walker, Deryck G.

doi:10.1042/bj1440149

Cited by 44 publications

(17 citation statements)

References 36 publications

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“…The results of the experiments described under Methods confirmed previously reported studies (16) indicating that the thyroid hormone-induced increase in ME activity was due to an increase in enzyme mass. The increment in the volume of antiserum required to neutralize 50% of the enzyme activity was indeed found to be proportional to the increment in enzyme activity induced by T3 administration (r = 0.93).…”

Section: Figure 1 Response Of Mitochondrial A-gpd (Upper Panel)supporting

confidence: 88%

“…Inhibitors of protein synthesis both at the transcriptional and translational levels block the enzyme response (6,8,9,13). The increase in enzyme activity appears to be a reflection of increased mass as demonstrated by the use of specific antisera to ME and measurement of increased rate of leuicine incorporation into this enzyme (16,18,20,22). Analogous studies with a-GPD have not been performed since specific antisera to this enzyme are not available.…”

Section: Abstract Experimentsmentioning

confidence: 99%

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Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Oppenheimer¹,

Silva²,

Schwartz³

et al. 1977

J. Clin. Invest.

153

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A B S T R A C T Experimentswere designed to analyze the relationship of a single i.v. dose of triiodothyronine (T3), the level of plasma and hepatic nuclear T3 attained, and the tissue response as reflected in increased activity of hepatic mitochondrial a-glycerophosphate dehydrogenase (a-GPD) and cytosol "malic enzyme" (ME). These studies were carried out in euthyroid rats by varying the dose of T3 injected and the time at which the animals were killed and the enzyme levels measured. The plasma T3 concentration was determined and the fraction of nuclear sites occupied at any time t was calculated from the known plasma:nuclear relationship. As a first step, the analysis was confined to the limiting situation in which all nuclear sites were effectively saturated. The following additional information was required and obtained: A proportional relationship between the half-neutralizing volume of a specific antiserum to malic enzyme and the activity of malic enzyme was established, thus confirming previous reports that the increase in enzyme activity induced by T3 is due to increased enzyme mass. The absolute refractory period immediately after i.v. injection of T3, during which no enzyme response could be detected, was determined. This was shown to be 13.4 h for a-GPD and 8.2 h for ME.Lastly, the tI of the enzyme decay after pulse injection of T3 was measured. This was similar for both (SD) days for ME.The results of these studies indicated that the extent of hepatic response appears limited by full occupancy of a set of intracellular receptor sites by T3 which is in rapid equilibrium with the plasma hormone pool. The kinetic properties of the receptors, as functionally defined in these studies, resemble those associated with the recently described specific nuclear T3 sites. These data per se are thus compatible with but do not prove a nuclear site of initiation of hormone effect. They do allow the development of an internally consistent mathematical model which permits prediction of enzyme response when the receptor sites are fully occupied for a given length of time after the i.v. injection of hormone.A separate series of studies was carried out in thyroidectomized rats. The response characteristics of a-GPD were similar to those observed in euthyroid animals. In contrast, however, the early response of ME to pulse injections of T3 was very much reduced in hypothyroid animals as compared to euthyroid animals in which nuclear sites were saturated for comparable periods. These findings raise the possibility that a factor required for the induction of malic enzyme but not a-GPD is deficient in the hypothyroid state.

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Section: Figure 1 Response Of Mitochondrial A-gpd (Upper Panel)supporting

confidence: 88%

Section: Abstract Experimentsmentioning

confidence: 99%

Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Oppenheimer¹,

Silva²,

Schwartz³

et al. 1977

J. Clin. Invest.

153

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show abstract

“…to the increment over hypothyroid levels achieved in Lbove, the assumption that the activity of the euthyroid state. The value off is 14.6 for a-GPD Leasured is proportional to enzyme mass and 14.3 for ME, values even higher than those calcufied in the case of ME (11,(22)(23)(24)(25). The lated from the 7-day experiment with full nuclear satu- (20) the value forf would be 2.13 in a linear system.…”

Section: Resultsmentioning

confidence: 82%

Nonlinear (amplified) relationship between nuclear occupancy by triiodothyronine and the appearance rate of hepatic alpha-glycerophosphate dehydrogenase and malic enzyme in the rat.

Oppenheimer¹,

Schwartz²,

Gutfeld³

1978

J. Clin. Invest.

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A B S T R A C T Three separate approaches were applied to examine the general relationship between R, the rate of induction of specific enzymes (mitochondrial a-glycerophosphate dehydrogenase and cytosolic malic enzyme) and q, the fractional nuclear occupancy by triiodothyronine in male Sprague-Dawley rats. Daily 200-,ug injections of triiodothyronine per 100 g body wt for 7 days resulted in saturation of the hepatic nuclear sites and the achievement of an apparent new steady state of enzyme levels. The increase achieved over base-line hypothyroid levels was then compared with the increment over hypothyroid base line characteristic of intact euthyroid animals with 47% of nuclear sites occupied. The maximal theoretical rate of steadystate enzyme induction could be projected on the basis of the observed maximal increase in enzyme activity observed 1 day after the injection of graded doses of hormone and X, the known fractional rate of enzyme dissipation. The 24-h dose-response studies were used to generate R as a continuous function of q, both in hypothyroid as well as in euthyroid animals. This approach involved the numerical solution of an ordinary differential equation describing the rate of change of enzyme as a function of R, which was assumed to be uniquely related to q. Results of these analyses indicated that the ratio of the maximal rate of induction

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“…This suggested these SH groups could move from 14 A to as close as 7 A upon binding of MgADP. We have recently provided further support for the proximal distance between SH-1 and SH-2 of [3][4][5] A based on evidence of simultaneous chelation of these two SH groups by a single-exchange-inert Co(III) phenanthroline complex (13,14).…”

mentioning

confidence: 99%

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

Wells¹,

Yount²

1979

Proc. Natl. Acad. Sci. U.S.A.

146

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Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of I'4CIATP or 114CIADP in a manner that depends linearly on the extent of inactivation by either NN'-p-phenylenedimaleimide or Co(II)phenanthroline/ICo(IIIXphenanthroline)2CO31+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 -5 days). The nucleotide incorporated is not covalently bound because HC104 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the 'y-phosphate of ly-32PJATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-#,B,y-imidoltriphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with NN'-p-phenylenedimaleimide or cobalt phenanthroline complexes. There is a large body of information available indicating that the myosin molecule and its active proteolytic subfragments, double-headed heavy meromyosin and single-headed subfragment 1 (SF-1), undergo spectroscopically sensitive conformational changes during ATP binding, hydrolysis, and product release (refs. 1-5; see ref. 6 for review). Binding of MgADP results in a well-documented (7-10) increase in the reactivity of one of two critical SH groups, so-called SH-2, suggesting this thiol moves from a buried to an exposed position.Reisler and coworkers (11) have reported that these critical thiols, SH-1 and SH-2, can be crosslinked by a 12-to 14-A thiol crosslinking reagent, N,N'-p-phenylenedimaleimide (pPDM). Furthermore, Burke and Reisler (12) reported that crosslinking with shorter bifunctional thiol reagents occurred optimally when the crosslinking was performed in the presence of MgADP. This suggested these SH groups could move from 14 A to as close as 7 A upon binding of MgADP. We have recently provided further support for the proximal distance between SH-1 and SH-2 of 3-5 A based on evidence of simultaneous chelation of these two SH groups by a single-exchange-inert Co(III) phenanthroline complex (13,14).Inactivations with pPDM (11) or cobalt phenanthroline complexes (13) are greatly stimulated by the addition of MgADP. Surprisingly, as shown here, adenosine 5-[t,-yimidoltriphosphate (AdoPP[NHIP) does not bind to SF-1 inactivated in the presence of MgADP with either reagent. These apparently conflicting observations are explained by the finding that nucleotide is trapped at the active site during nucleotidestimulated inactivation. We provide evidence that the nucleotide is specifically trapped at the hydrolytic site and is only slowly released over a period of days. The trapped nucleotide is rapidly released when the enzyme deriva...

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Enzyme synthesis in the regulation of hepatic ‘malic’ enzyme activity

Cited by 44 publications

References 36 publications

Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Nonlinear (amplified) relationship between nuclear occupancy by triiodothyronine and the appearance rate of hepatic alpha-glycerophosphate dehydrogenase and malic enzyme in the rat.

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

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