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The regulatory kinetic properties of porcine hepatic glucokinase

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Abstract

Porcine hepatic glucokinase (ATP: D-hexose 6-phosphotransferase EC 2.7.1.1) has been purified by a modification of the procedure for its purification from rats. However, difficulties were encountered with endogenous proteases and the reliability of a source for porcine livers. The molecular weight has been determined to be 60 400 ± 1400 by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. The enzyme has been characterized kinetically. The parameter values, S 0.5 (glucose) and Hill coefficient (nH) are 2.4 mM and 1.9 respectively under sulfhydryl-reducing conditions. The enzyme undergoes the two sulfhydryl-related decays of its activity previously observed in the enzyme isolated from rat (Tippett PS, Neet KE: Arch Biochem Biophys 222:285–298, 1983). The enzyme is inhibited by palmitoyl-CoA, K i (apparent) = 1.0 µM, nH = 1.8; this concentration of inhibitor is significantly below its critical micelle concentration. Physically and kinetically glucokinase isolated from pig is similar to the enzyme isolated from rat. The porcine system provides a second source for isolation and further characterization of this important and unusual enzyme.

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Abbreviations

Bis-Tris-propane:

1,3-bis[tris-(hydroxymethyl)methylamino]propane

DTT:

Dithiothreitol

Ki :

Inhibitor Constant

nH :

Hill coefficient

PMSF:

Phenylmethanesulfonyl Fluoride

S0.5 :

Substrate concentration for half-maximal activity

SDS-PAGE:

Sodium Dodecyl Sulfate, Polyacrylamide gel electrophoresis

τ:

tau, the reciprocal of the rate constant for the transition from the initial to the steady-state velocity. V i, V ss, and V max, Initial, Steady-State and Maximal Velocities

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Author notes

  1. Walter K. Vogel

    Present address: Department of Biochemistry and Biophysics, Oregon State University, 97331-6503, Corvallis, Oregon, USA

Authors and Affiliations

  1. Department of Biochemistry, University Hospitals, Case Western Reserve University School of Medicine, 44106, Cleveland, Ohio, USA

    Walter K. Vogel, Robert P. Keenan & Kenneth E. Neet

  2. Department of Medicines, University Hospitals Case Western Reserve University, School of Medicine, 44106, Cleveland, Ohio, USA

    Christina W. Gelev

Authors
  1. Walter K. Vogel
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  2. Robert P. Keenan
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  3. Christina W. Gelev
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  4. Kenneth E. Neet
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Additional information

A portion of this work is taken from a thesis submitted by W. K. Vogel to Case Western Reserve University in partial fulfillment of the requirements for the degree of Master of Science.

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Vogel, W.K., Keenan, R.P., Gelev, C.W. et al. The regulatory kinetic properties of porcine hepatic glucokinase. Mol Cell Biochem 86, 171–179 (1989). https://doi.org/10.1007/BF00222617

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  • DOI: https://doi.org/10.1007/BF00222617

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