The kinetic consequences of binding of hexokinase-I to the mitochondrial outer membrane

F.D. Laterveer, Ron Heijden, van der, M. Toonen, K. Nicolaij

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Abstract

In a number of tissues, a major fraction of the hexokinase isozyme species present is bound at the mitochondrial outer surface. This study addresses the kinetic consequences of binding of hexokinase to the outer membrane of isolated, phosphorylating mitochondria. The primary aim was to separately measure the relative contributions to changes in the kinetic properties of hexokinase which (1) directly result from the binding as such, and (2) are caused by binding in close proximity to the site of mitochondrial ATP regeneration. Hexokinase isozyme I was purified from rat brain and then bound to intact rat liver mitochondria or outer membrane vesicles derived from these mitochondria. The apparent affinity (Km app) for ATP and the Vmax of the bound hexokinase were determined by the spectrophotometric measurement of its activity as a function of the ATP concentration in the medium. The data obtained for the bound enzyme in the two systems were compared to the kinetic characteristics of hexokinase-I present in a non-bound form. Non-bindable hexokinase was obtained by mild protease treatment, such that bindability was completely abolished while the intrinsic catalytic properties remained unaltered. Parallel determinations of the steady-state ATP and ADP levels in mitochondrial suspensions with bound or non-bindable hexokinase present provided additional information on the consequences of binding. Binding of hexokinase to phosphorylating mitochondria decreased the Km app for ATP from 0.168 to 0.081 mM while not changing the Vmax. It appeared that both binding per se (Km app for ATP decreased from 0.168 to 0.105 mM and from 0.194 to 0.103 mM upon binding to non-phosphorylating mitochondria and outer membrane vesicles, respectively) and intramitochondrial ATP regeneration (causing a further reduction in Km app for ATP from 0.105 to 0.081 mM for the system of phosphorylating mitochondria) jointly contributed to this reduction in Km app for ATP caused by binding to phosphorylating mitochondria. The kinetic effect exerted by intramitochondrial ATP regeneration persisted in the presence of an excess of extramitochondrial ATP regenerating activity. ATP and ADP measurements in hexokinase-mitochondria incubations demonstrated that: (i) up to 7 mM ATP in the medium, higher ADP concentrations were maintained for the case of non-bindable enzyme as compared to the bound enzyme; and (ii) ATP levels were not significantly different and therefore not responsible for the kinetic difference between bound and non-bound hexokinase. The present findings are compatible with previous suggestions for local channelling of adenine nucleotides between bound hexokinase and oxidative phosphorylation.
Original languageEnglish
Pages (from-to)251-259
Number of pages9
JournalBiochimica et Biophysica Acta, Bioenergetics
Volume1188
Issue number3
DOIs
Publication statusPublished - 1994

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Hexokinase
Mitochondrial Membranes
Adenosine Triphosphate
Mitochondria
Membranes
Kinetics
Application programs
Adenosine Diphosphate
Regeneration
Isoenzymes
Rats
Enzymes
Adenine Nucleotides
Liver Mitochondrion
Oxidative Phosphorylation
Liver

Cite this

Laterveer, F.D. ; Heijden, van der, Ron ; Toonen, M. ; Nicolaij, K. / The kinetic consequences of binding of hexokinase-I to the mitochondrial outer membrane. In: Biochimica et Biophysica Acta, Bioenergetics. 1994 ; Vol. 1188, No. 3. pp. 251-259.
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abstract = "In a number of tissues, a major fraction of the hexokinase isozyme species present is bound at the mitochondrial outer surface. This study addresses the kinetic consequences of binding of hexokinase to the outer membrane of isolated, phosphorylating mitochondria. The primary aim was to separately measure the relative contributions to changes in the kinetic properties of hexokinase which (1) directly result from the binding as such, and (2) are caused by binding in close proximity to the site of mitochondrial ATP regeneration. Hexokinase isozyme I was purified from rat brain and then bound to intact rat liver mitochondria or outer membrane vesicles derived from these mitochondria. The apparent affinity (Km app) for ATP and the Vmax of the bound hexokinase were determined by the spectrophotometric measurement of its activity as a function of the ATP concentration in the medium. The data obtained for the bound enzyme in the two systems were compared to the kinetic characteristics of hexokinase-I present in a non-bound form. Non-bindable hexokinase was obtained by mild protease treatment, such that bindability was completely abolished while the intrinsic catalytic properties remained unaltered. Parallel determinations of the steady-state ATP and ADP levels in mitochondrial suspensions with bound or non-bindable hexokinase present provided additional information on the consequences of binding. Binding of hexokinase to phosphorylating mitochondria decreased the Km app for ATP from 0.168 to 0.081 mM while not changing the Vmax. It appeared that both binding per se (Km app for ATP decreased from 0.168 to 0.105 mM and from 0.194 to 0.103 mM upon binding to non-phosphorylating mitochondria and outer membrane vesicles, respectively) and intramitochondrial ATP regeneration (causing a further reduction in Km app for ATP from 0.105 to 0.081 mM for the system of phosphorylating mitochondria) jointly contributed to this reduction in Km app for ATP caused by binding to phosphorylating mitochondria. The kinetic effect exerted by intramitochondrial ATP regeneration persisted in the presence of an excess of extramitochondrial ATP regenerating activity. ATP and ADP measurements in hexokinase-mitochondria incubations demonstrated that: (i) up to 7 mM ATP in the medium, higher ADP concentrations were maintained for the case of non-bindable enzyme as compared to the bound enzyme; and (ii) ATP levels were not significantly different and therefore not responsible for the kinetic difference between bound and non-bound hexokinase. The present findings are compatible with previous suggestions for local channelling of adenine nucleotides between bound hexokinase and oxidative phosphorylation.",
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The kinetic consequences of binding of hexokinase-I to the mitochondrial outer membrane. / Laterveer, F.D.; Heijden, van der, Ron; Toonen, M.; Nicolaij, K.

In: Biochimica et Biophysica Acta, Bioenergetics, Vol. 1188, No. 3, 1994, p. 251-259.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - The kinetic consequences of binding of hexokinase-I to the mitochondrial outer membrane

AU - Laterveer, F.D.

AU - Heijden, van der, Ron

AU - Toonen, M.

AU - Nicolaij, K.

PY - 1994

Y1 - 1994

N2 - In a number of tissues, a major fraction of the hexokinase isozyme species present is bound at the mitochondrial outer surface. This study addresses the kinetic consequences of binding of hexokinase to the outer membrane of isolated, phosphorylating mitochondria. The primary aim was to separately measure the relative contributions to changes in the kinetic properties of hexokinase which (1) directly result from the binding as such, and (2) are caused by binding in close proximity to the site of mitochondrial ATP regeneration. Hexokinase isozyme I was purified from rat brain and then bound to intact rat liver mitochondria or outer membrane vesicles derived from these mitochondria. The apparent affinity (Km app) for ATP and the Vmax of the bound hexokinase were determined by the spectrophotometric measurement of its activity as a function of the ATP concentration in the medium. The data obtained for the bound enzyme in the two systems were compared to the kinetic characteristics of hexokinase-I present in a non-bound form. Non-bindable hexokinase was obtained by mild protease treatment, such that bindability was completely abolished while the intrinsic catalytic properties remained unaltered. Parallel determinations of the steady-state ATP and ADP levels in mitochondrial suspensions with bound or non-bindable hexokinase present provided additional information on the consequences of binding. Binding of hexokinase to phosphorylating mitochondria decreased the Km app for ATP from 0.168 to 0.081 mM while not changing the Vmax. It appeared that both binding per se (Km app for ATP decreased from 0.168 to 0.105 mM and from 0.194 to 0.103 mM upon binding to non-phosphorylating mitochondria and outer membrane vesicles, respectively) and intramitochondrial ATP regeneration (causing a further reduction in Km app for ATP from 0.105 to 0.081 mM for the system of phosphorylating mitochondria) jointly contributed to this reduction in Km app for ATP caused by binding to phosphorylating mitochondria. The kinetic effect exerted by intramitochondrial ATP regeneration persisted in the presence of an excess of extramitochondrial ATP regenerating activity. ATP and ADP measurements in hexokinase-mitochondria incubations demonstrated that: (i) up to 7 mM ATP in the medium, higher ADP concentrations were maintained for the case of non-bindable enzyme as compared to the bound enzyme; and (ii) ATP levels were not significantly different and therefore not responsible for the kinetic difference between bound and non-bound hexokinase. The present findings are compatible with previous suggestions for local channelling of adenine nucleotides between bound hexokinase and oxidative phosphorylation.

AB - In a number of tissues, a major fraction of the hexokinase isozyme species present is bound at the mitochondrial outer surface. This study addresses the kinetic consequences of binding of hexokinase to the outer membrane of isolated, phosphorylating mitochondria. The primary aim was to separately measure the relative contributions to changes in the kinetic properties of hexokinase which (1) directly result from the binding as such, and (2) are caused by binding in close proximity to the site of mitochondrial ATP regeneration. Hexokinase isozyme I was purified from rat brain and then bound to intact rat liver mitochondria or outer membrane vesicles derived from these mitochondria. The apparent affinity (Km app) for ATP and the Vmax of the bound hexokinase were determined by the spectrophotometric measurement of its activity as a function of the ATP concentration in the medium. The data obtained for the bound enzyme in the two systems were compared to the kinetic characteristics of hexokinase-I present in a non-bound form. Non-bindable hexokinase was obtained by mild protease treatment, such that bindability was completely abolished while the intrinsic catalytic properties remained unaltered. Parallel determinations of the steady-state ATP and ADP levels in mitochondrial suspensions with bound or non-bindable hexokinase present provided additional information on the consequences of binding. Binding of hexokinase to phosphorylating mitochondria decreased the Km app for ATP from 0.168 to 0.081 mM while not changing the Vmax. It appeared that both binding per se (Km app for ATP decreased from 0.168 to 0.105 mM and from 0.194 to 0.103 mM upon binding to non-phosphorylating mitochondria and outer membrane vesicles, respectively) and intramitochondrial ATP regeneration (causing a further reduction in Km app for ATP from 0.105 to 0.081 mM for the system of phosphorylating mitochondria) jointly contributed to this reduction in Km app for ATP caused by binding to phosphorylating mitochondria. The kinetic effect exerted by intramitochondrial ATP regeneration persisted in the presence of an excess of extramitochondrial ATP regenerating activity. ATP and ADP measurements in hexokinase-mitochondria incubations demonstrated that: (i) up to 7 mM ATP in the medium, higher ADP concentrations were maintained for the case of non-bindable enzyme as compared to the bound enzyme; and (ii) ATP levels were not significantly different and therefore not responsible for the kinetic difference between bound and non-bound hexokinase. The present findings are compatible with previous suggestions for local channelling of adenine nucleotides between bound hexokinase and oxidative phosphorylation.

U2 - 10.1016/0005-2728(94)90043-4

DO - 10.1016/0005-2728(94)90043-4

M3 - Article

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EP - 259

JO - Biochimica et Biophysica Acta, Bioenergetics

JF - Biochimica et Biophysica Acta, Bioenergetics

SN - 0005-2728

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ER -