Studies on the reaction mechanism of soybean callus succinyl CoA synthetase. phosphorylation of the enzyme on immobilized adenosine‐triphosphate and enzyme attached to a solid support

Xifra, Eva A.Wider De; Mendiara, S. N.; Batlle, Alcira

doi:10.1016/0014-5793(72)80639-2

Cited by 19 publications

(6 citation statements)

References 19 publications

(2 reference statements)

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“…In most cases, the immobilized nucleotides have been used as a step in enzyme purification (8,(20)(21)(22)(23)(24)(25). They have also been used as characterization tools for studies such as the phosphorylation of succinyl CoA synthetase (24) and the interaction between myosin species and ATP (25).…”

Section: Discussionmentioning

confidence: 99%

Binding of ATP to the progesterone receptor.

Moudgil¹,

Toft²

1975

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

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The An initial step in the mechanism of steroid hormone action involves binding of the steroid to receptor proteins in the target cell. The resulting complex then migrates into the cell nucleus and is thought to act as a modulator of gene expression (for review, see refs. 1-3). However, the actual biochemical function of the receptor remains unknown. This is the case because little is known about the regulation of gene activity in higher organisms, and also, because of difficulties in the purification and characterization of the receptor proteins. Some insight into the function of steroid receptors might be gained by studying their ability to interact with various cellular constituents. This approach has been used with the avian progesterone receptor to demonstrate its ability to bind to DNA and chromatin (4). Possible interactions between the receptor and other cellular constituents are difficult to analyze, particularly if the ligand is of a common type and of low molecular weight. Highly purified receptor preparations are not yet available for direct binding studies.We have used affinity chromatography as a relatively unambiguous and direct procedure for measuring the possible interaction of progesterone receptors with various nucleotides. While a role of nucleotides in the initial steps of hormone action is not directly evident, previous studies (5-7) indicate: (i) that energy-requiring or enzymatic steps may be involved in activation of the hormone-receptor complex and its translocation to nuclear sites of actions, and (ii) that this nuclear action is closely associated with the process of RNA synthesis. In addition, there have been a few reports that implicate a relationship between steroid receptors and ATP (see Discussion).The present results show a specific binding between the progesterone receptor and ATP, suggesting an involvement of this nucleotide in some aspect of receptor function. MATERIALS AND METHODSAll reagents were of analytical grade and were made up in glass-distilled water. All ATP was oxidized with sodium metaperiodate according to the method of Gilham (10). To 10 ml of Sepharose-hydrazide preparation in 0.1 M sodium acetate buffer (pH 5) were added 70 ,umol of periodate-oxidized ATP in a total volume of 25 ml of 0.1 M\I sodium acetate, pH 5.0 (8). The suspension was placed in a shaker for 3 hr at 40, after which 2 M NaCl (75 ml) was added and shaking was continued for another 30 min. This suspension was filtered under reduced pressure and washed with 1.0 liter of distilled water and 10Q ml of TETG 901 Abbreviation: TETG, 50 mM Tris-HCl, 1 mM EDTA, 12 mM monothioglycerol (pH 8).

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Section: Discussionmentioning

confidence: 99%

Binding of ATP to the progesterone receptor.

Moudgil¹,

Toft²

1975

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

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“…With enzyme preparations phosphorylated with either ATP or 32P-ATP and Mg2+, under conditions described previously [3], it was found that the enzyme is quite stable upon precipitation with ammonium sul phate and after repeated filtration through Sephadex G-50 columns.…”

Section: Stability Of the Phosphoryl Enzymementioning

confidence: 99%

“…Attachment of the enzyme to Sepharose 4B, preparation of the phosphoryl en zyme from both soluble and insolubilized Suc-CoA-S, determination of proteinbound phosphate and phosphates, as well as all other materials and methods not specified here, were those described earlier [3]; 0.05 m tris-HCl buffer pH 8.0 was used throughout. The gel filtration technique was always used to isolate various complexes, such as those of the enzyme-bound phosphate, enzyme-bound CoA, or the enzyme-bound phosphate-CoA.…”

Section: Methodsmentioning

confidence: 99%

“…An In experiment 1 the Seph-E-P was prepared by incubating Seph-E with ATP and Mg2+ as described previously [3], In experiment 2 prior to phosphorylation with ATP and Mg2+ carried out in the same way as in experiment 1, the Seph-E packed in a column was treated with CoA and Mg2+, as described in the text. Afterwards, catalytic activity of the different preparations was determined.…”

Section: ]mentioning

confidence: 99%

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Mechanism of Action of Succinyl CoA Synthetase

Xifra¹,

Am²

1973

Enzyme

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“…Mawal et al [S] have confirmed the presence of galactosyltransferase-substrate complexes, predicted from steady-state kinetic data, by investigating the interaction ofthe enzyme with a-lactalbuminSepharose. Similarly, by immobilising either the enzyme or ATP, the mechanism of succinyl-CoA synthetase phosphorylation has been successfully examined [7]. The catalytic and regulatory subunits of kinases dependent on adenosine 3' : Ei'-monophosphate have been studied using casein-Sepharose ; the results demonstrated that 3' : 5'-AMP activates protein kinases by the dissociation of an enzymeinhibitor complex [8].…”

mentioning

confidence: 99%

Affinity Chromatography on Immobilised Adenosine 5'-monophosphate. Some Kinetic Parameters Involved in the Binding of Group-Specific Enzymes

Lowe¹,

Harvey²,

Dean³

1974

Eur J Biochem

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The interaction of several enzymes with N6-(6-aminohexyl)-5'-AMP-Xepharose was examined under column chromatographic and batchwise conditions. The dissociation constant ( K L ) of lactate dehydrogenase and glycerokinase for immobilised 5'-AMP was 1.1 pM when determined under batchwise conditions ; similar values were recorded for lactate dehydrogenase in the presence of a competitive inhibitor (NADH). The effect of competitive idubitors on the binding of alcohol dehydrogenase, glycerokinase and lactate dehydrogenase to Ng-(6-aminohexyl)-5'-AiiP-Sepharose under column chromatographic conditions was studied. Under these conditions the dissociation constant (KL) of lactate dehydrogenase for the immobilised 5'-AMP was about 0.27 nM, the strength of the interaction (p) decreased with increasing NADH concentrations and a relationship was apparent between the elution volume and inhibitor concentration. These results have been discussed in relation to the principles of afsnity chromatography, and are correlated with the known kinetic and molecular properties of the enzymes in free solution.The application of biospecific adsorbents in the isolation and purification of enzymes is now a wellestablished procedure [l, 21. Furthermore, several reports indicate that a qualitative evaluation of complex interactions can be achieved by immobilising one component of the system and studying the binding parameters of the interacting species. Thus Akanuma et al. [5] has been examined with various amino acid-Sepharose columns and correlated with the free solution kinetics of the enzymes. Mawal et al. [S] have confirmed the presence of galactosyltransferase-substrate complexes, predicted from steady-state kinetic data, by investigating the interaction ofthe enzyme with a-lactalbuminSepharose. Similarly, by immobilising either the enzyme or ATP, the mechanism of succinyl-CoA synthetase phosphorylation has been successfully examined [7]. The catalytic and regulatory subunits of kinases dependent on adenosine 3' : Ei'-monophosphate have been studied using casein-Sepharose ; the results demonstrated that 3' : 5'-AMP activates protein kinases by the dissociation of an enzymeinhibitor complex [8]. Gawronski and Wold [9] Enzymes. Lactate dehydrogenase (EC 1.1.1.27); alcohol dehydrogenase (EC 1 A1.1) ; glycerokinase (EC 2.7.1.30).studying the ribonuclease &' -protein -&peptide interaction examined the feasibility of obtaining quantitative binding data for the interaction when either of the components was immobilised to agarose. The dissociation constants obtained by dwect binding studies were virtually independent of the immobilised component and were within the published range of dissociation constants for this interaction.I n the present investigation the kinetic paremeters of the interaction of group-specific enzymes with N6-(6-aminohexyl)-5'-AMP-Sepharose, with particular reference to lactate dehydrogenase, have been examined. The results have been interpreted in the knowledge of the documented behaviour of the enzymes in free solutio...

show abstract

Studies on the reaction mechanism of soybean callus succinyl CoA synthetase. phosphorylation of the enzyme on immobilized adenosine‐triphosphate and enzyme attached to a solid support

Cited by 19 publications

References 19 publications

Binding of ATP to the progesterone receptor.

Binding of ATP to the progesterone receptor.

Mechanism of Action of Succinyl CoA Synthetase

Affinity Chromatography on Immobilised Adenosine 5'-monophosphate. Some Kinetic Parameters Involved in the Binding of Group-Specific Enzymes

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