Sigmoidal Kinetics of Glucokinase

Niemeyer, Hermann M.; M, de la Luz Cárdenas; Rabajille, Eliana; Ureta, Tito; Clark-Turri, Lyllian; Peñaranda, J. Benezet

doi:10.1159/000458957

Cited by 73 publications

(45 citation statements)

References 8 publications

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“…In the absence of regulatory protein, both enzymes displayed a sigmoidal saturation curve for glucose, with Hill coefficients of 1.57 and 1.6 for the rat and the Xenopus enzymes, respectively. The So, value was notably smaller for Xenopus (1.9 mM) compared to the rat glucokinase (8.1 mM), as was previously shown for the enzyme from other amphibian species (Niemeyer et al, 1975). The native and the recombinant regulatory proteins had the same effect on the kinetics of glucokinases; they both behaved as competitive inhibitors with respect to glucose, and did not affect V,,,, as indicated by double-reciprocal plots (not shown).…”

Section: Characterisation Of the Recombinant Regulatory Protein And Csupporting

confidence: 74%

Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase

Veiga‐da‐Cunha¹,

Detheux²,

Watelet³

et al. 1994

European Journal of Biochemistry

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Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1 -phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551 -5561. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucolunase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.The liver of the rat contains a regulatory protein, also called regulator, which inhibits glucokinase competitively with respect to glucose . Fructose 6-phosphate and fructose 1-phosphate are ligands of this regulatory protein and act competitively, fructose 6-phosphate to reinforce the inhibition, and fructose 1-phosphate to antagonize it. In addition, C1-and other monovalent anions reduce the inhibition exerted by the regulatory protein noncompetitively with respect to fructose 6-phosphate. The presence of the regulatory protein in rat liver explains the activating effect of fructose (Van Schaftingen and Vandercammen, 1989;Davies et al., 1990) and of a potassium-rich medium on the phosphorylation of glucose in isolated hepatocytes. The cDNA encoding the rat liver regulatory protein has been recently cloned (Detheux et al., 1993).A fructose-6-phosphate-sensitive and fructose-l-phosphate-sensitive regulatory protein appears to be present in the livers of all mammalian species that express hepatic glucokinase, including man (Vandercammen and Van Schaftingen, 1993). The livers of the amphibians Bufo marinus and Xenopus laevis, and of the turtle Pseudemys scripta elegans con- tain a protein that inhibits glucokinase competitively with respect to glucose but is insensitive to fructose 6-phosphate and fructose 1-phosphate (Vandercammen and Van Schaftingen, 1993). It was of interest to know if this form of regulatory protein of glucokinase was homologous to ...

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Section: Characterisation Of the Recombinant Regulatory Protein And Csupporting

confidence: 74%

Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase

Veiga‐da‐Cunha¹,

Detheux²,

Watelet³

et al. 1994

European Journal of Biochemistry

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“…It has been known for a long time [l I] that the hexokinases from Saccharomyces and other microorganisms [15,49] are not inhibited by glucose-6-P and that fact may be important as a criterion for studying the evolutionary relationships of the glucose : ATP phosphotransferases. The fungus hexokinases differ from the isozymes C and D because vertebrate hexokinase C is inhibited by excess glucose [25] and hexokinase D, or glucokinase, presents a more restricted substrate specificity, sigmoidal kinetics and a relative molecular mass of about 50000 [13,27,461.…”

Section: Discussionmentioning

confidence: 99%

“…The lowest K , value for glucose was 0.056 mM (hexokinase p) and the highest, 0.18 mM (hexokinase 6). None of the isozymes presented inhibition by excess glucose as in the case of vertebrate hexokinase C [25,26] or sigmoidicity as observed with vertebrate hexokinase D [27,28]. Likewise, none of the N .…”

Section: Characterization Of the Hexokinases From Neurospora Crassamentioning

confidence: 99%

The Hexokinases from Wild-Type and Morphological Mutant Strains of Neurospora crassa

Lagos

Ureta

1980

Eur J Biochem

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Four hexose-phosphorylating isozymes (hexokinases a, B, y and 6) were separated by DEAEcellulose column chromatography of high-speed supernatants from Neurospora crassa wild-type strains 77a and RL3-8A. The isozymes displayed broad sugar specificity and Michaelis constants for glucose in the micromolar range. The isozymes were not inhibited by glucose 6-phosphate nor activated by citrate. Molecular weights were estimated as 95 000 by gel filtration. Glucose-specific kinases were not found.Marked quantitative variations in the relative proportion of the hexokinases were observed during growth of the fungus on minimal medium plus sucrose. Hexokinase p was the predominant form (82% of the total activity) during the period of hyphal growth. At maturation time all the isozymes were present at about the same level although a small preponderance of hexokinase fl was still evident.The relative proportions of the hexokinases did not vary when sucrose was replaced by glucose, fructose or casamino acids. When sorbose was added to the minimal medium containing sucrose, a high proportion of hexokinase p was apparent concomitantly with the well known change of growth to a colonial mode. Several morphological mutant strains were examined with respect to the hexokinase profile.Strains COI-16 and crisp showed a marked preponderance of hexokinase p. Extracts from the dapple and dingy strains lacked hexokinases a and 6. Rabbit antisera to commercial preparations of hexokinase PI and PI1 from Saccharomyces cerevisiae were able to inhibit partially the enzyme activity of Neurospora hexokinases y and 6.No effect of the antisera on hexokinases a and B could be observed.Multiple hexokinases were first recognized in the yeast Succhuromyces cerevisiae in 1961 by Trayser and Colowick [l] and by several other research groups afterwards [2,3]. The existence of two hexokinases of broad sugar specificities, designated as PI and PI1 (or A and B), and a rather specific glucokinase, seems now well established. Hexokinases PI and PI1 have been thoroughly studied by a number of investigators with respect to kinetic properties [4] and structural features [5] ; the elucidation of the amino acid sequence of one of the isozymes may be possible soon. Recent detailed genetic studies by Maitra and his group [6,7] and by Fraenkel and coworkers [8] have shown that

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“…So far, the only exception is the observation of a limited degree of dimerization occurring in aged preparations of partially purified glucokinase, the dimer exhibiting hyperbolic kinetics (3). This monomeric structure of glucokinase is of special interest since the enzyme displays kinetic properties such that the velocity as a function of glucose concentration shows sigmoidal dependence (3,4). In view of the monomeric nature of glucokinase, interpretation of the sigmoidicity becomes of considerable theoretical importance.…”

Section: Discussionmentioning

confidence: 99%

“…However, only recently it has become apparent that the saturation function of glucokinase for glucose does not follow the classical Michaelian hyperbole, but exhibits a sigmoidal shape instead (3,4). The Hill coefficient (no) is about 1.6 for the glucokinases of all vertebrates so far examined (3).…”

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confidence: 99%