The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium. The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme. There is one site for each of these ligands per monomer. In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, i.e., all homotropic interactions of the substrate are lost. In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator. The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator. The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer. In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer. Magnesium ions inhibit binding of GDP and GTP. KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc., enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate. The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other.

The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified. The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography. On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000. Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme. The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions. Sulfate and phosphate ions are inhibitory to the enzyme. Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators. The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway. Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators. Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu. Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation. The effectors act by changing the Michaelis constant for the substrates. Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate. The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.

Abstract. To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with ot-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pmod(I) and proot2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.

The D-lactate oxidation dependent transport of succinate in membrane vesicles of an Escherichia coli strain lacking succinate dehydrogenase and fumarate reductase is inhibited by several categories of compounds. One category consists of compounds that are electron transport inhibitors (Amytal, Dicumarol, and mercurials), the second of compounds that act as competitive inhibitors of D-lactate dehydrogenase (oxamate and β-chlorolactate), the third of reagents that inhibit the Ca2+–Mg2+-activated ATPase (dicyclohexylcarbodiimide and pyrophosphate), and the fourth of compounds that tap off electrons from the respiratory chain (2,6-dichlorophenolindophenol). None of the succinate transport inhibitors, including mercurials like p-chloromercuribenzoate, interfere with the binding of succinate to the presumed membrane carriers.Membrane preparations from mutants of E. coli lacking D-lactate dehydrogenase are unable to transport succinate in the presence of D-lactate. Whole cells of these mutants, however, take up succinate normally. This observation suggests that D-lactate oxidation is not obligatorily linked in vivo to the uptake of succinate although the possibility is not excluded that transport in such mutants may be linked to some other dehydrogenase. Mutants having altered levels of ATPase, or membrane preparations made from such cells also have greatly reduced capacity to transport succinate. This observation coupled with the finding that ATPase inhibitors block dicarboxylate transport suggests involvement of ATPase in an unknown way in the concentrative uptake of succinate.With the exception of oxamate, β-chlorolactate (competitive inhibitors of D-lactate oxidation), and dicyclohexylcarbodiimide, all of the inhibitors of succinate uptake (including p-chloromercuribenzoate) cause an immediate efflux of preloaded succinate from membrane vesicles. Efflux is also caused by proton conducting reagents. The Km for efflux is 1.9 mM. This value is to be compared with the Km for influx, which is only about 0.02 mM.The weight of evidence favors the view that the active transport of succinate in vesicles occurs as a result of an energization of the membranes by the passage of electrons, although alternate oxidation and reduction of the succinate carrier as a mechanism for transport has not been definitely ruled out.

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